ANTIGEN BINDING MOLECULES WITH INCREASED Fc RECEPTOR BINDING AFFINITY AND EFFECTOR FUNCTION

ABSTRACT

The present invention relates to antigen binding molecules (ABMs). In particular embodiments, the present invention relates to recombinant monoclonal antibodies, including chimeric, primatized or humanized antibodies specific for human CD20. In addition, the present invention relates to nucleic acid molecules encoding such ABMs, and vectors and host cells comprising such nucleic acid molecules. The invention further relates to methods for producing the ABMs of the invention, and to methods of using these ABMs in treatment of disease. In addition, the present invention relates to ABMs with modified glycosylation having improved therapeutic properties, including antibodies with increased Fc receptor binding and increased effector function.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/981,738, filed Nov. 5, 2004, which claims the benefit of U.S.Provisional Application No. 60/517,096, filed Nov. 5, 2003, thedisclosures of which are herein incorporated by reference in theirentirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392023112SeqList.txt,date recorded: Jun. 3, 2015, size: 58 KB).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to antigen binding molecules (ABMs). Inparticular embodiments, the present invention relates to recombinantmonoclonal antibodies, including chimeric, primatized or humanizedantibodies specific for human CD20. In addition, the present inventionrelates to nucleic acid molecules encoding such ABMs, and vectors andhost cells comprising such nucleic acid molecules. The invention furtherrelates to methods for producing the ABMs of the invention, and tomethods of using these ABMs in treatment of disease. In addition, thepresent invention relates to ABMs with modified glycosylation havingimproved therapeutic properties, including antibodies with increased Fcreceptor binding and increased effector function.

2. Background Art

The Immune System and Anti-CD20 Antibodies

The immune system of vertebrates, including humans, consists of a numberof organs and cell types, which have evolved to accurately andspecifically recognize, bind and destroy invading foreign microorganisms(“antigens”). Lymphocytes are critical for the proper function of theimmune system. These cells are produced in the thymus, spleen and bonemarrow (adult) and represent about 30% of the total white blood cellspresent in the circulatory system of adult humans. There are two majorsub-populations of lymphocytes: T cells and B cells. T cells areresponsible for cell mediated immunity, while B cells are responsiblefor antibody production (humoral immunity). However, in a typical immuneresponse, T cells and B cells function interdependently: T cells areactivated when the T cell receptor binds to fragments of an antigen thatare bound to major histocompatability complex (“MHC”) glycoproteins onthe surface of an antigen presenting cell; such activation causesrelease of biological mediators (“interleukins”), which stimulate Bcells to differentiate and produce antibodies (“immunoglobulins”)against the antigen.

Each B cell within the host expresses an antibody of one particular typeand specificity, and different B cells express antibodies specific fordifferent antigens. B cell proliferation and antibody production spikeas a reaction to a foreign antigen, and both typically cease (orsubstantially decrease) once the foreign antigen has been neutralized.Occasionally, however, proliferation of a particular B cell willcontinue unabated; such proliferation can result in a cancer referred toas “B cell lymphoma.”

T cells and B cells both comprise cell surface proteins which can beutilized as “markers” for differentiation and identification. One suchhuman B cell marker is the human B lymphocyte-restricted differentiationantigen Bp35, referred to as “CD20.” CD20 is expressed during earlypre-B cell development and remains until plasma cell differentiation.Specifically, the CD20 molecule may regulate a step in the activationprocess that is required for cell cycle initiation and differentiationand is usually expressed at very high levels on neoplastic (“tumor”) Bcells. Because CD20 is present at high levels on “malignant” B cells,i.e., those B cells whose unabated proliferation can lead to B celllymphoma, the CD20 surface antigen has the potential of serving as acandidate for “targeting” of B cell lymphomas.

In essence, such targeting can be generalized as follows: antibodiesspecific to the CD20 surface antigen of B cells are introduced into apatient, by injection, for example. These anti-CD20 antibodiesspecifically bind to the CD20 cell surface antigen of (ostensibly) bothnormal and malignant B cells; the anti-CD20 antibody bound to the CD20surface antigen may lead to the destruction and depletion of neoplasticB cells. Additionally, chemical agents or radioactive labels having thepotential to destroy the tumor can be conjugated to the anti-CD20antibody such that the agent is specifically “delivered” to e.g., theneoplastic B cells. Irrespective of the approach, a primary goal is todestroy the tumor: the specific approach can be determined by theparticular anti-CD20 antibody which is utilized and, thus, the availableapproaches to targeting the CD20 antigen can vary considerably.

Unconjugated monoclonal antibodies (mAbs) can be useful medicines forthe treatment of cancer, as demonstrated by the U.S. Food and DrugAdministration's approval of Rituximab (Rituxan™; IDEC Pharmaceuticals,San Diego, Calif., and Genentech Inc., San Francisco, Calif.), for thetreatment of CD20 positive B-cell, low-grade or follicular Non-Hodgkin'slymphoma, Trastuzumab (Herceptin™; Genentech Inc,) for the treatment ofadvanced breast cancer (Grillo-Lopez, A.-J., et al., Semin. Oncol.26:66-73 (1999); Goldenberg, M. M., Clin. Ther. 21:309-18 (1999)),Gemtuzumab (Mylotarg™, Celltech/Wyeth-Ayerst) for the treatment ofrelapsed acute myeloid leukemia, and Alemtuzumab (CAMPATH™, MilleniumPharmaceuticals/Schering AG) for the treatment of B cell chroniclymphocytic leukemia. The success of these products relies not only ontheir efficacy but also on their outstanding safety profiles(Grillo-Lopez, A.-J., et al., Semin. Oncol. 26:66-73 (1999); Goldenberg,M. M., Clin. Ther. 21:309-18 (1999)). In spite of the achievements ofthese drugs, there is currently a large interest in obtaining higherspecific antibody activity than what is typically afforded byunconjugated mAb therapy. The murine monoclonal antibody, B-Ly1, isanother antibody known to be specific to human CD20. (Poppema, S. andVisser, L., Biotest Bulletin 3: 131-139 (1987)).

The results of a number of studies suggest that Fc-receptor-dependentmechanisms contribute substantially to the action of cytotoxicantibodies against tumors and indicate that an optimal antibody againsttumors would bind preferentially to activation Fc receptors andminimally to the inhibitory partner FcγRIIB. (Clynes, R. A., et al.,Nature Medicine 6(4):443-446 (2000); Kalergis, A. M., and Ravetch, J.V., J. Exp. Med. 195(12):1653-1659 (June 2002). For example, the resultsof at least one study suggest that the FcγRIIIa receptor in particularis strongly associated with the efficacy of antibody therapy. (Cartron,G., et al., Blood 99(3):754-757 (February 2002)). That study showed thatpatients homozygous for FcγRIIIa have a better response to Rituximabthan heterozygous patients. The authors concluded that the superiorresponse was due to better in vivo binding of the antibody to FcγRIIIa,which resulted in better ADCC activity against lymphoma cells. (Cartron,G., et al., Blood 99(3):754-757 (February 2002)).

Various attempts to target the CD20 surface antigen have been reported.Murine (mouse) monoclonal antibody IF5 (an anti-CD20 antibody) wasreportedly administered by continuous intravenous infusion to B celllymphoma patients. Extremely high levels (>2 grams) of 1F5 werereportedly required to deplete circulating tumor cells, and the resultswere described as being “transient.” Press et al., “Monoclonal Antibody1F5 (Anti-CD20) Serotherapy of Human B-Cell Lymphomas.” Blood69/2:584-591 (1987). A potential problem with this approach is thatnon-human monoclonal antibodies (e.g., murine monoclonal antibodies)typically lack human effector functionality, i.e., they are unable to,inter alia, mediate complement dependent lysis or lyse human targetcells through antibody dependent cellular toxicity or Fc-receptormediated phagocytosis. Furthermore, non-human monoclonal antibodies canbe recognized by the human host as a foreign protein; therefore,repeated injections of such foreign antibodies can lead to the inductionof immune responses leading to harmful hypersensitivity reactions. Formurine-based monoclonal antibodies, this is often referred to as a HumanAnti-Mouse Antibody response, or “HAMA” response. Additionally, these“foreign” antibodies can be attacked by the immune system of the hostsuch that they are, in effect, neutralized before they reach theirtarget site.

Another reported approach at improving the ability of murine monoclonalantibodies to be effective in the treatment of B-cell disorders has beento conjugate a radioactive label or toxin to the antibody such that thelabel or toxin is localized at the tumor site. For example, theabove-referenced 1F5 antibody has been “labeled” with iodine-131(“¹³¹I”) and was reportedly evaluated for biodistribution in twopatients. See Eary, J. F. et al., “Imaging and Treatment of B-CellLymphoma” J. Nuc. Med. 31/8:1257-1268 (1990); see also. Press, O. W. etal., “Treatment of Refractory Non-Hodgkin's Lymphoma with RadiolabeledMB-1 (Anti-CD37) Antibody” J. Clin. Onc. 7/8:1027-1038 (1989)(indication that one patient treated with ¹³¹I-labeled IF-5 achieved a“partial response”); Goldenberg, D. M. et al., “Targeting, Dosimetry andRadioinmmunotherapy of B-Cell Lymphomas with Iodine-131-Labeled LL2Monoclonal Antibody” J. Clin. Onc. 9/4:548-564 (1991) (three of eightpatients receiving multiple injections reported to have developed a HAMAresponse); Appelbaum, F. R. “Radiolabeled Monoclonal Antibodies in theTreatment of Non-Hodgkin's Lymphoma” Hem./Onc. Clinics of N. A.5/5:1013-1025 (1991) (review article); Press, O. W. et al“Radiolabeled-Antibody Therapy of B-Cell Lymphoma with Autologous BoneMarrow Support.” New England J. Med. 329/17: 1219-12223 (1993)(iodine-131 labeled anti-CD20 antibody IF5 and B1); and Kaminski, M. G.et al “Radioimmunotherapy of B-Cell Lymphoma with ¹³¹I Anti-B1(Anti-CD20) Antibody”. New England J. Med. 329/7(1993) (iodine-131labeled anti-CD20 antibody B1; hereinafter “Kaminski”). Toxins (i.e.,chemotherapeutic agents such as doxorubicin or mitomycin C) have alsobeen conjugated to antibodies. See, for example, PCT publishedapplication WO 92/07466 (published May 14, 1992).

Chimeric antibodies comprising portions of antibodies from two or moredifferent species (e.g., mouse and human) have been developed as analternative to “conjugated” antibodies. For example, Liu, A. Y. et al,“Production of a Mouse-Human Chimeric Monoclonal Antibody to CD20 withPotent Fc-Dependent Biologic Activity” J. Immun. 139/10:3521-3526(1987), describes a mouse/human chimeric antibody directed against theCD20 antigen. See also, PCT Publication No. WO 88/04936. For example,rituximab (RITUXAN®), a chimeric anti-CD20, antibody has been approvedfor the treatment of non-Hodgkins lymphoma.

Given the expression of CD20 by B cell lymphomas, this antigen can serveas a candidate for “targeting” of such lymphomas. In essence, suchtargeting can be generalized as follows: antibodies specific for CD20surface antigen on B cells are administered to a patient. Theseanti-CD20 antibodies specifically bind to the CD20 antigen of(ostensibly) both normal and malignant B cells, and the antibody boundto the CD20 on the cell surface results in the destruction and depletionof tumorigenic B cells. Additionally, chemical agents, cytotoxins orradioactive agents may be directly or indirectly attached to theanti-CD20 antibody such that the agent is selectively “delivered” to theCD20 antigen expressing B cells. With both of these approaches, theprimary goal is to destroy the tumor. The specific approach will dependupon the particular anti-CD20 antibody that is utilized. Thus, it isapparent that the various approaches for targeting the CD20 antigen canvary considerably.

The rituximab (RITUXAN®) antibody is a genetically engineered chimerichuman gamma 1 murine constant domain containing monoclonal antibodydirected against the human CD20 antigen. This chimeric antibody containshuman gamma 1 constant domains and is identified by the name “C2B8” inU.S. Pat. No. 5,736,137 (Andersen et. al.) issued on Apr. 17, 1998,assigned to IDEC Pharmaceuticals Corporation. RITUXAN® is approved forthe treatment of patients with relapsed or refracting low-grade orfollicular, CD20 positive, B cell non-Hodgkin's lymphoma. In vitromechanism of action studies have shown that RITUXAN® exhibits humancomplement-dependent cytotoxicity (CDC) (Reff et. al, Blood 83(2):435-445 (1994)). Additionally, it exhibits significant activity inassays that measure antibody-dependent cellular cytotoxicity (ADCC).RITUXAN® has been shown to possess anti-proliferative activity inthymidine incorporation assays and a limited ability to induce apoptosisdirectly, whereas CD20 antibodies do not (Maloney et. al, Blood 88 (10):637a (1996)).

Antibody Glycosylation

The oligosaccharide component can significantly affect propertiesrelevant to the efficacy of a therapeutic glycoprotein, includingphysical stability, resistance to protease attack, interactions with theimmune system, pharmacokinetics, and specific biological activity. Suchproperties may depend not only on the presence or absence, but also onthe specific structures, of oligosaccharides. Some generalizationsbetween oligosaccharide structure and glycoprotein function can be made.For example, certain oligosaccharide structures mediate rapid clearanceof the glycoprotein from the bloodstream through interactions withspecific carbohydrate binding proteins, while others can be bound byantibodies and trigger undesired immune reactions. (Jenkins et al.,Nature Biotechnol. 14:975-81 (1996)).

Mammalian cells are the preferred hosts for production of therapeuticglycoproteins, due to their capability to glycosylate proteins in themost compatible form for human application. (Cumming et al.,Glycobiology 1:115-30 (1991); Jenkins et al., Nature Biotechnol.14:975-81 (1996)). Bacteria very rarely glycosylate proteins, and likeother types of common hosts, such as yeasts, filamentous fungi, insectand plant cells, yield glycosylation patterns associated with rapidclearance from the blood stream, undesirable immune interactions, and insome specific cases, reduced biological activity. Among mammalian cells,Chinese hamster ovary (CHO) cells have been most commonly used duringthe last two decades. In addition to giving suitable glycosylationpatterns, these cells allow consistent generation of genetically stable,highly productive clonal cell lines. They can be cultured to highdensities in simple bioreactors using serum-free media, and permit thedevelopment of safe and reproducible bioprocesses. Other commonly usedanimal cells include baby hamster kidney (BHK) cells, NS0- andSP2/0-mouse myeloma cells. More recently, production from transgenicanimals has also been tested. (Jenkins et al., Nature Biotechnol.14:975-81 (1996)).

All antibodies contain carbohydrate structures at conserved positions inthe heavy chain constant regions, with each isotype possessing adistinct array of N-linked carbohydrate structures, which variablyaffect protein assembly, secretion or functional activity. (Wright, A.,and Morrison, S. L., Trends Biotech. 15:26-32 (1997)). The structure ofthe attached N-linked carbohydrate varies considerably, depending on thedegree of processing, and can include high-mannose, multiply-branched aswell as biantennary complex oligosaccharides. (Wright, A., and Morrison,S. L., Trends Biotech. 15:26-32 (1997)). Typically, there isheterogeneous processing of the core oligosaccharide structures attachedat a particular glycosylation site such that even monoclonal antibodiesexist as multiple glycoforms. Likewise, it has been shown that majordifferences in antibody glycosylation occur between cell lines, and evenminor differences are seen for a given cell line grown under differentculture conditions. (Lifely, M. R. et al., Glycobiology 5(8):813-22(1995)).

One way to obtain large increases in potency, while maintaining a simpleproduction process and potentially avoiding significant, undesirableside effects, is to enhance the natural, cell-mediated effectorfunctions of monoclonal antibodies by engineering their oligosaccharidecomponent as described in Umaña, P. et al., Nature Biotechnol.17:176-180 (1999) and U.S. Pat. No. 6,602,684, the entire contents ofwhich are hereby incorporated by reference in their entirety. IgG1 typeantibodies, the most commonly used antibodies in cancer immunotherapy,are glycoproteins that have a conserved N-linked glycosylation site atAsn297 in each CH2 domain. The two complex biantennary oligosaccharidesattached to Asn297 are buried between the CH2 domains, forming extensivecontacts with the polypeptide backbone, and their presence is essentialfor the antibody to mediate effector functions such as antibodydependent cellular cytotoxicity (ADCC) (Lifely, M. R., et al.,Glycobiology 5:813-822 (1995); Jefferis, R., et al., Immunol Rev.163:59-76 (1998); Wright, A. and Morrison, S. L., Trends Biotechnol.15:26-32 (1997)).

The present inventors showed previously that overexpression in Chinesehamster ovary (CHO) cells of β(1,4)-N-acetylglucosaminyltransferase III(“GnTIII”), a glycosyltransferase catalyzing the formation of bisectedoligosaccharides, significantly increases the in vitro ADCC activity ofan anti-neuroblastoma chimeric monoclonal antibody (chCE7) produced bythe engineered CHO cells. (See Umaña, P. et al., Nature Biotechnol.17:176-180 (1999); and International Publication No. WO 99/54342, theentire contents of which are hereby incorporated by reference). Theantibody chCE7 belongs to a large class of unconjugated mAbs which havehigh tumor affinity and specificity, but have too little potency to beclinically useful when produced in standard industrial cell lineslacking the GnTIII enzyme (Umana, P., et al., Nature Biotechnol.17:176-180 (1999)). That study was the first to show that largeincreases of ADCC activity could be obtained by engineering theantibody-producing cells to express GnTIII, which also led to anincrease in the proportion of constant region (Fc)-associated, bisectedoligosaccharides, including bisected, nonfucosylated oligosaccharides,above the levels found in naturally-occurring antibodies.

There remains a need for enhanced therapeutic approaches targeting theCD20 antigen for the treatment of B cell lymphomas in primates,including, but not limited to, humans.

BRIEF SUMMARY OF THE INVENTION

Recognizing the tremendous therapeutic potential of antigen bindingmolecules (ABMs) that have the binding specificity of the murine B-Ly1antibody and that have been glycoengineered to enhance Fe receptorbinding affinity and effector function, the present inventors developeda method for producing such ABMs. Inter alia, this method involvesproducing recombinant, chimeric antibodies or chimeric fragmentsthereof. The efficacy of these ABMs is further enhanced by engineeringthe glycosylation profile of the antibody Fc region.

Accordingly, in one aspect, the invention is directed to an isolatedpolynucleotide comprising: (a) a sequence selected from a groupconsisting of: SEQ ID NO.:5, SEQ ID NO.:6 and SEQ ID NO.:7. (CDRsV_(H-1)); (b) a sequence selected from a group consisting of: SEQ IDNO.:21, SEQ ID NO.:22 and SEQ ID NO.:23, (CDRs V_(H-2)); and SEQ IDNO:24. In another aspect, the invention is directed to an isolatedpolynucleotide comprising SEQ ID NO.:8, SEQ ID NO.:9 and SEQ ID NO.:10,(CDRs V_(L)). In one embodiment, any of these polynucleotides encodes afusion polypeptide.

In a further aspect, the invention is directed to an isolatedpolynucleotide comprising SEQ ID No.: 3 In another aspect, the inventionis directed to an isolated polynucleotide comprising SEQ ID No.: 4. In afurther aspect, the invention is directed to an isolated polynucleotidecomprising a sequence selected from the group consisting of SEQ IDNo:29; SEQ ID No:31; SEQ ID No:33; SEQ ID No:35; SEQ ID No:37; SEQ IDNo:39; SEQ ID No:41; SEQ ID No:43; SEQ ID No:45; SEQ ID No:47; SEQ IDNo:49; SEQ ID No:51; SEQ ID No:53; SEQ ID No:55; SEQ ID No:57; SEQ IDNo:59; SEQ ID No:61; SEQ ID No:63; SEQ ID No:65; SEQ ID No:67; SEQ IDNo:69; and SEQ ID No:71. In another aspect, the invention is directed toan isolated polynucleotide comprising SEQ ID No.: 75. In one embodiment,such polynucleotides encode fusion polypeptides.

The invention is further directed to an isolated polynucleotidecomprising a sequence having at least 80% identity to SEQ ID NO:3,wherein said isolated polynucleotide encodes a fusion polypeptide. In anadditional aspect, the invention is directed to an isolatedpolynucleotide comprising a sequence having at least 80% identity to SEQID NO:4, wherein said isolated polynucleotide encodes a fusionpolypeptide. The invention is further directed to an isolatedpolynucleotide comprising a sequence having at least 80% identity to asequence selected from the group consisting of SEQ ID No:29; SEQ IDNo:31; SEQ ID No:33; SEQ ID No:35; SEQ ID No:37; SEQ ID No:39; SEQ IDNo:41; SEQ ID No:43; SEQ ID No:45; SEQ ID No:47; SEQ ID No:49; SEQ IDNo:51; SEQ ID No:53; SEQ ID No:55; SEQ ID No:57; SEQ ID No:59; SEQ IDNo:61; SEQ ID No:63; SEQ ID No:65; SEQ ID No:67; SEQ ID No:69; and SEQID No:71, wherein said isolated polynucleotide encodes a fusionpolypeptide. In an additional aspect, the invention is directed to anisolated polynucleotide comprising a sequence having at least 80%identity to SEQ ID NO:75, wherein said isolated polynucleotide encodes afusion polypeptide.

The invention is further directed to a polynucleotide comprising SEQ IDNO: 11 (whole heavy chain), or to polynucleotides having 80%, 85%, 90%,95% or 99% identity to SEQ ID NO:11. The invention is also directed to apolynucleotide comprising SEQ ID NO:12 (whole light chain), or topolynucleotides having 80%, 85%, 90%, 95% or 99% identity to SEQ ID NO:12.

The invention is also directed to an isolated polynucleotide encoding achimeric polypeptide having the sequence of SEQ ID No.: 1. In oneembodiment, the polynucleotide comprises a sequence encoding apolypeptide having the sequence of SEQ ID No.: 1; and a sequenceencoding a polypeptide having the sequence of an antibody Fe region, ora fragment thereof, from a species other than mouse. The invention isalso directed to an isolated polynucleotide encoding a chimericpolypeptide having a sequence selected from the group consisting of SEQID No:30; SEQ ID No:32; SEQ ID No:34; SEQ ID No:36; SEQ ID No:38; SEQ IDNo:40; SEQ ID No:42; SEQ ID No:44; SEQ ID No:46; SEQ ID No:48; SEQ IDNo:50; SEQ ID No:52; SEQ ID No:54; SEQ ID No:56; SEQ ID No:58; SEQ IDNo:60; SEQ ID No:62; SEQ ID No:64; SEQ ID No:66; SEQ ID No:68; SEQ IDNo:70; and SEQ ID No:72. In one embodiment, the polynucleotide comprisesa sequence encoding a polypeptide having a sequence selected from thegroup consisting of SEQ ID No:30; SEQ ID No:32; SEQ ID No:34; SEQ IDNo:36; SEQ ID No:38; SEQ ID No:40; SEQ ID No:42; SEQ ID No:44; SEQ IDNo:46; SEQ ID No:48; SEQ ID No:50; SEQ ID No:52; SEQ ID No:54; SEQ IDNo:56; SEQ ID No:58; SEQ ID No:60; SEQ ID No:62; SEQ ID No:64; SEQ IDNo:66; SEQ ID No:68; SEQ ID No:70; and SEQ ID No:72; and a sequenceencoding a polypeptide having the sequence of an antibody Fc region, ora fragment thereof, from a species other than mouse.

In yet another aspect, the invention is directed to an isolatedpolynucleotide encoding a chimeric polypeptide having the sequence ofSEQ ID No.: 2. In one embodiment, the polynucleotide comprises asequence encoding a polypeptide having the sequence of SEQ ID No.: 2;and a sequence encoding a polypeptide having the sequence of an antibodyFc region, or a fragment thereof, from a species other than mouse. Inyet another aspect, the invention is directed to an isolatedpolynucleotide encoding a chimeric polypeptide having the sequence ofSEQ ID No.: 76. In one embodiment, the polynucleotide comprises asequence encoding a polypeptide having the sequence of SEQ ID No.: 76;and a sequence encoding a polypeptide having the sequence of an antibodyFc region, or a fragment thereof, from a species other than mouse.

The invention is also directed to an isolated polynucleotide comprisinga sequence encoding a polypeptide having the V_(H) region of the murineB-Ly1 antibody, or functional variants thereof, and a sequence encodinga polypeptide having the sequence of an antibody Fc region, or afragment thereof, from a species other than mouse. In another aspect,the invention is directed to an isolated polynucleotide comprising asequence encoding a polypeptide having the V_(L) region of the murineB-Ly1 antibody, or functional variants thereof, and a sequence encodinga polypeptide having the sequence of an antibody Fe region, or afragment thereof, from a species other than mouse.

The invention is further directed to an expression vector comprising anyof the isolated polynucleotides described above, and to a host cell thatcomprises such an expression vector. In a further aspect, the inventionis directed to a host cell comprising any of the isolatedpolynucleotides described above.

In one aspect, the invention is directed to an isolated polypeptidecomprising: (a) a sequence selected from a group consisting of: SEQ IDNO.:15, SEQ ID NO.:16 and SEQ ID NO.:17. (CDRs V_(H-1)); (b) a sequenceselected from a group consisting of: SEQ ID NO.:25, SEQ ID NO.:26 andSEQ ID NO.:27 (CDRs V_(H-2)); and SEQ ID NO:28, wherein said polypeptideis a fusion polypeptide. In another aspect, the invention is directed toan isolated polypeptide comprising SEQ ID NO.:18, SEQ ID NO.:19 and SEQID NO.:20. (CDRs V_(L)) wherein said polypeptide is a fusionpolypeptide.

The invention is also directed to a chimeric polypeptide comprising thesequence of SEQ ID NO.:1 or a variant thereof. The invention is furtherdirected to a chimeric polypeptide comprising the sequence of SEQ IDNO.:2 or a variant thereof. In one embodiment, any one of thesepolypeptides further comprises a human Fc region. The invention is alsodirected to a chimeric polypeptide comprising a sequence selected fromthe group consisting of SEQ ID No:30; SEQ ID No:32; SEQ ID No:34; SEQ IDNo:36; SEQ ID No:38; SEQ ID No:40; SEQ ID No:42; SEQ ID No:44; SEQ IDNo:46; SEQ ID No:48; SEQ ID No:50; SEQ ID No:52; SEQ ID No:54; SEQ IDNo:56; SEQ ID No:58; SEQ ID No:60; SEQ ID No:62; SEQ ID No:64; SEQ IDNo:66; SEQ ID No:68; SEQ ID No:70; and SEQ ID No:72, or a variantthereof. The invention is further directed to a chimeric polypeptidecomprising the sequence of SEQ ID NO.:76 or a variant thereof. In oneembodiment, any one of these polypeptides further comprises a human Fcregion.

In another aspect the invention is directed to a polypeptide comprisinga sequence derived from the murine B-Ly1 antibody and a sequence derivedfrom a heterologous polypeptide and to an antigen-binding moleculecomprising such a polypeptide. In one embodiment the antigen-bindingmolecule is an antibody. In a preferred embodiment, the antibody ischimeric. In another preferred embodiment, the antibody is humanized orprimatized.

In an additional aspect, the invention is directed to an isolatedpolypeptide comprising SEQ ID NO: 13 or a variant thereof. In anotheraspect, the invention is directed to an isolated polypeptide comprisingSEQ ID NO: 14.

In another aspect, the invention is directed to an ABM, which is capableof competing with the murine B-Ly1 antibody for binding to CD20 andwhich is chimeric. In one embodiment, the ABM is an antibody or afragment thereof. In a further embodiment, the ABM is a recombinantantibody comprising a V_(H) region having an amino acid sequenceselected from the group consisting of SEQ ID NO.:1; SEQ ID No:30; SEQ IDNo:32; SEQ ID No:34; SEQ ID No:36; SEQ ID No:38; SEQ ID No:40; SEQ IDNo:42; SEQ ID No:44; SEQ ID No:46; SEQ ID No:48; SEQ ID No:50; SEQ IDNo:52; SEQ ID No:54; SEQ ID No:56; SEQ II) No:58; SEQ ID No:60; SEQ IDNo:62; SEQ ID No:64; SEQ ID No:66; SEQ ID No:68; SEQ ID No:70; and SEQID No:72. In another embodiment, the ABM is a recombinant antibodycomprising a V_(L) region having an amino acid sequence selected fromthe group consisting of SEQ ID NO.:2 and SEQ ID NO:76. In a furtherembodiment the ABM is a recombinant antibody that is primatized. In yeta further embodiment the ABM is a recombinant antibody that ishumanized. In another embodiment, the ABM is a recombinant antibodycomprising a human Fc region. In a further embodiment, any of the ABMsdiscussed above may be conjugated to a moiety such as a toxin or aradiolabel.

The invention is further related to an ABM of the present invention,said ABM having modified oligosaccharides. In one embodiment themodified oligosaccharides have reduced fucosylation as compared tonon-modified oligosaccharides. In other embodiments, the modifiedoligosaccharides are hybrid or complex. In a further embodiment, the ABMhas an increased proportion of nonfucosylated oligosaccharides orbisected, nonfucosylated oligosaccharides in the Fc region of saidmolecule. In one embodiment, the bisected, nonfucosylatedoligosaccharides are hybrid. In a further embodiment, the bisected,nonfucosylated oligosaccharides are complex. In a one embodiment, atleast 20% of the oligosaccharides in the Fc region of said polypeptideare nonfucosylated or bisected, nonfucosylated. In more preferredembodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65/%, 70% or75% or more of the oligosaccharides are nonfucosylated or bisected,nonfucosylated.

The invention is further related to a polynucleotide encoding any of theABMs discussed above, and to expression vectors and cells comprisingsuch a polynucleotide.

The invention is further related to a method of producing an ABM, whichis capable of competing with the murine B-Ly1 antibody for binding toCD20 and wherein said ABM is chimeric; said method comprising: (a)culturing a host cell comprising a polynucleotide that encodes an ABM ofthe present invention in a medium under conditions allowing theexpression of said polynucleotide encoding said ABM; and (b) recoveringsaid ABM from the resultant culture.

In another aspect, the invention is related to a pharmaceuticalcomposition comprising the ABM of the invention. It is contemplated thatthe pharmaceutical composition may further comprise a pharmaceuticallyacceptable carrier, an adjuvant or a combination thereof.

In a further aspect, the invention is related to a method of treating adisease treatable by B-cell depiction. The method comprisesadministering a therapeutically effective amount of the ABM of thepresent invention to a human subject in need thereof. In a preferredembodiment, the disease is treated by administering an ABM that is achimeric antibody, or a chimeric fragment of an antibody.

In yet another aspect, the invention is related to a host cellengineered to express at least one nucleic acid encoding a polypeptidehaving GnTIII activity in an amount sufficient to modify theoligosaccharides in the Fc region of produced by the host cell, whereinthe ABM is capable of competing with the murine B-Ly1 antibody forbinding to CD20 and wherein the ABM is chimeric. In one embodiment, thepolypeptide having GnTIII activity is a fusion polypeptide. In anotherembodiment, the ABM produced by the host cell is an antibody or anantibody fragment. In a further embodiment, the ABM comprises a regionequivalent to the Fc region of a human IgG.

The invention is also directed to an isolated polynucleotide comprisingat least one complementarity determining region of the murine B-Ly1antibody, or a variant or truncated form thereof containing at least thespecificity-determining residues for said complementarity determiningregion, wherein said isolated polynucleotide encodes a fusionpolypeptide. Preferably, such isolated polynucleotides encode a fusionpolypeptide that is an antigen binding molecule. In one embodiment, thepolynucleotide comprises three complementarity determining regions ofthe murine B-Ly1 antibody, or variants or truncated forms thereofcontaining at least the specificity-determining residues for each ofsaid three complementarity determining regions. In another embodiment,the polynucleotide encodes the entire variable region of the light orheavy chain of a chimeric (e.g., humanized) antibody. The invention isfurther directed to the polypeptides encoded by such polynucleotides.

In another embodiment, the invention is directed to an antigen combiningmolecule comprising at least one complementarity determining region ofthe murine B-Ly1 antibody, or a variant or truncated form thereofcontaining at lest the specificity-determining residues for saidcomplementarity determining region, and comprising a sequence derivedfrom a heterologous polypeptide. In one embodiment, the antigen bindingmolecule comprises three complementarity determining regions of themurine B-Ly1 antibody, or variants or truncated forms thereof containingat least the specificity-determining residues for each of said threecomplementarity determining regions. In another aspect, the antigenbinding molecule comprises the variable region of an antibody light orheavy chain. In one particularly useful embodiment, the antigen bindingmolecule is a chimeric, e.g., humanized, antibody. The invention is alsodirected to methods of making such antigen binding molecules, and theuse of same in the treatment of disease, including B cell lymphomas.

The present invention is the first known instance in which a Type IIanti-CD20 antibody has been engineered to have increases effectorfunctions such as ADCC, while still retaining potent apoptosis ability.Accordingly, the present invention is directed to an engineered Type IIanti-CD20 antibody having increased ADCC as a result of said engineeringand without loss of substantial ability to induces apoptosis. In oneembodiment, the Type II anti-CD20 antibodies have been engineered tohave an altered pattern of glycosylation in the Fc region. In aparticular embodiment, the altered glycosylation comprises an increasedlevel of bisected complex residues in the Fc region. In anotherparticular embodiment, the altered glycosylation comprises and reducedlevel of fucose residues in the Fc region. In another embodiment, theType II anti-CD20 antibodies have undergone polypeptide engineering. heinvention is further directed to methods of making such engineered TypeII antibodies and to methods of using such antibodies in the treatmentof various B cell disorders, including B cell lymphomas.

The host cell of the present invention may be selected from the groupthat includes, but is not limited to, a CHO cell, a BHK cell, a NSOcell, a SP2/0 cell, a YO myeloma cell, a P3X63 mouse myeloma cell, a PERcell, a PER.C6 cell or a hybridoma cell. In one embodiment, the hostcell of the invention further comprises a transfected polynucleotidecomprising a polynucleotide encoding the V_(L) region of the murineB-Ly1 antibody or variants thereof and a sequence encoding a regionequivalent to the Fc region of a human immunoglobulin. In anotherembodiment, the host cell of the invention further comprises atransfected polynucleotide comprising a polynucleotide encoding theV_(H) region of the murine B-Ly1 antibody or variants thereof and asequence encoding a region equivalent to the Fc region of a humanimmunoglobulin.

In a further aspect, the invention is directed to a host cell thatproduces an ABM that exhibits increased Fc receptor binding affinityand/or increased effector function as a result of the modification ofits oligosaccharides. In one embodiment, the increased binding affinityis to an Fc receptor, particularly, the FcγRIIIA receptor. The effectorfunction contemplated herein may be selected from the group thatincludes, but is not limited to, increased Fc-mediated cellularcytotoxicity; increased binding to NK cells; increased binding tomacrophages; increased binding to polymorphonuclear cells; increasedbinding to monocytes; increased direct signaling inducing apoptosis;increased dendritic cell maturation; and increased T cell priming.

In a further embodiment, the host cell of the present inventioncomprises at least one nucleic acid encoding a polypeptide having GnTIIIactivity that is operably linked to a constitutive promoter element.

In another aspect, the invention is directed to a method for producingan ABM in a host cell, comprising: (a) culturing a host cell engineeredto express at least one polynucleotide encoding a fusion polypeptidehaving GnTIII activity under conditions which permit the production ofsaid ABM and which permit the modification of the oligosaccharidespresent on the Fc region of said ABM; and (b) isolating said ABM;wherein said ABM is capable of competing with the murine B-Ly1 antibodyfor binding to CD20 and wherein said ABM is chimeric. In one embodiment,the polypeptide having GnTIII activity is a fusion polypeptide,preferably comprising the catalytic domain of GnTIII and the Golgilocalization domain of a heterologous Golgi resident polypeptideselected from the group consisting of the localization domain ofmannosidase II, the localization domain ofβ(1,2)-N-acetylglucosaminyltransferase I (“GnTI”), the localizationdomain of mannosidase I, the localization domain ofβ(1,2)-N-acetylglucosaminyltransferase II (“GnTII”), and thelocalization domain of α1-6 core fucosyltransferase. Preferably, theGolgi localization domain is from mannosidase II or GnTI.

In a further aspect, the invention is directed to a method for modifyingthe glycosylation profile of an anti-CD20 ABM produced by a host cellcomprising introducing into the host cell at least one nucleic acid orexpression vector of the invention. In one embodiment, the ABM is anantibody or a fragment thereof; preferably comprising the Fc region ofan IgG. Alternatively, the polypeptide is a fusion protein that includesa region equivalent to the Fc region of a human IgG.

In one aspect, the invention is related to a recombinant, chimericantibody, or a fragment thereof, capable of competing with the murineB-Ly1 antibody for binding to CD20 and having reduced fucosylation.

In another aspect, the present invention is directed to a method ofmodifying the glycosylation of the recombinant antibody or a fragmentthereof of the invention by using a fusion polypeptide having GnTIIIactivity and comprising the Golgi localization domain of a heterologousGolgi resident polypeptide. In one embodiment, the fusion polypeptidesof the invention comprise the catalytic domain of GnTIII. In anotherembodiment, the Golgi localization domain is selected from the groupconsisting of: the localization domain of mannosidase II, thelocalization domain of GnTI, the localization domain of mannosidase I,the localization domain of GnTII and the localization domain of α1-6core fucosyltransferase. Preferably, the Golgi localization domain isfrom mannosidase II or GnTI.

In one embodiment, the method of the invention is directed towardsproducing a recombinant, chimeric antibody or a fragment thereof, withmodified oligosaccharides wherein said modified oligosaccharides havereduced fucosylation as compared to non-modified oligosaccharides.According to the present invention, these modified oligosaccharides maybe hybrid or complex. In another embodiment, the method of the inventionis directed towards producing a recombinant, chimeric antibody or afragment thereof having an increased proportion of bisected,nonfucosylated oligosaccharides in the Fc region of said polypeptide. Inone embodiment, the bisected, nonfucosylated oligosaccharides arehybrid. In another embodiment, the bisected, nonfucosylatedoligosaccharides are complex. In a further embodiment, the method of theinvention is directed towards producing a recombinant, chimeric antibodyor a fragment thereof having at least 20% of the oligosaccharides in theFc region of said polypeptide that are bisected, nonfucosylated. In apreferred embodiment, at least 30% of the oligosaccharides in the Fcregion of said polypeptide are bisected, nonfucosylated. In anotherpreferred embodiment, wherein at least 35% of the oligosaccharides inthe Fc region of said polypeptide are bisected, nonfucosylated.

In a further aspect, the invention is directed to a recombinant,chimeric antibody or a fragment thereof, that exhibits increased Fcreceptor binding affinity and/or increased effector function as a resultof the modification of its oligosaccharides. In one embodiment, theincreased binding affinity is to an Fc activating receptor. In a furtherembodiment, the Fc receptor is Fey activating receptor, particularly,the FcγRIIIA receptor. The effector function contemplated herein may beselected from the group that includes, but is not limited to, increasedFc-mediated cellular cytotoxicity; increased binding to NK cells;increased binding to macrophages; increased binding to polymorphonuclearcells; increased binding to monocytes; increased direct signalinginducing apoptosis; increased dendritic cell maturation; and increased Tcell priming.

In another aspect, the invention is directed to a recombinant, chimericantibody fragment, having the binding specificity of the murine B-Ly1antibody and containing the Fc region, that is engineered to haveincreased effector function produced by any of the methods of thepresent invention.

In another aspect, the present invention is directed to a fusion proteinthat includes a polypeptide having the sequence of SEQ ID NO:1 and aregion equivalent to the Fc region of an immunoglobulin and engineeredto have increased effector function produced by any of the methods ofthe present invention.

In another aspect, the present invention is directed to a fusion proteinthat includes a polypeptide having the sequence of SEQ ID NO:2 and aregion equivalent to the Fc region of an immunoglobulin and engineeredto have increased effector function produced by any of the methods ofthe present invention.

In one aspect, the present invention is directed to a pharmaceuticalcomposition comprising a recombinant, chimeric antibody, produced by anyof the methods of the present invention, and a pharmaceuticallyacceptable carrier. In another aspect, the present invention is directedto a pharmaceutical composition comprising a recombinant, chimericantibody fragment produced by any of the methods of the presentinvention, and a pharmaceutically acceptable carrier. In another aspect,the present invention is directed to a pharmaceutical compositioncomprising a fusion protein produced by any of the methods of thepresent invention, and a pharmaceutically acceptable carrier.

The invention is further directed to a method of treating a diseasetreatable by B-cell depletion comprising administering a therapeuticallyeffective amount of the recombinant, chimeric, antibody or fragmentthereof, produced by any of the methods of the present invention, to ahuman subject in need thereof.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Nucleotide (SEQ ID NO:2), complimentary nucleotide (SEQ IDNO:85) and amino acid sequence (SEQ ID NO: 1) of the V_(H) region of themurine B-Ly1. Also shown are the nucleotide sequence of the murine B-Ly1V_(H) region linker (SEQ ID NO:79), the complementary nucleotidesequence (SEQ ID NO:80), and the amino acid sequence (SEQ ID NO:81).

FIG. 2. Nucleotide (SEQ ID NO:4), complimentary nucleotide (SEQ IDNO:86) and amino acid sequence (SEQ ID NO:3) of the V_(L) region of themurine B-Ly1. Also shown are the nucleotide sequence of the murine B-Ly1VL region linker (SEQ ID NO:82), the complementary nucleotide sequence(SEQ ID NO:83), and the amino acid sequence (SEQ ID NO:84).

FIG. 3. Binding of Rituximab® (O) and ch-B_Ly1 (Δ) to CD20 on RajiB-lymphoma cells.

FIG. 4A, 4B, 4C. B-Cell depletion by Rituximab® (O) and ch-B_Ly1 (Δ) inwhole blood of the three different classes of FcγRIIIa-158V/F genotype:(FIG. 4A) whole blood from a F/F donor, homozygous for the loweraffinity receptor; (FIG. 4B) whole blood from a F/V donor, heterozygousfor the affinity receptor; and (FIG. 4C) whole blood from a V/V donor,homozygous for the higher affinity receptor.

FIG. 5A, 5B, 5C. Nucleotide (SEQ ID NO: 11) and amino acid sequence (SEQID NO: 13) of the heavy chain of a chimeric, anti-CD20 antibody.

FIGS. 6A and 6B. Nucleotide (SEQ ID NO:12) and amino acid sequence (SEQID NO: 14) of the light chain of a chimeric, anti-CD20 antibody.

FIGS. 7A and 7B. Nucleotide and amino acid sequences of the murine B-Ly1antibody CDRs. (FIG. 7A) Predicted CDRs for the V_(H) region. (FIG. 7B)Predicted CDRs for the V_(L) region.

FIG. 8A, 8B, 8C. MALDI-TOF profile of a glycoengineered, chimeric B-Ly1antibody. (FIG. 8A) Table detailing the percentages of specific peaks;(FIG. 8B) Spectrum for glycoengineered chimeric B-Ly1; (FIG. 8C)Spectrum for glycoengineered chimeric B-Ly1 treated with Endo-H.

FIG. 9. Binding of different humanized anti-CD20 antibodies to RajiB-cells. The differences between the B-HH2 construct and the B-HL8 andB-HL11 constructs are located in the framework 1 and 2 regions with allthree CDRs being identical. B-HL8 and B-HL11 have their FR1 and FR2sequences derived from the human VH3 class, whereas the complete B-HH2framework is human VH1 derived. B-HL11 is a derivative of B-HL8 with thesingle mutation Glu1Gln, with Gln being the amino acid residue in theB-HH2 construct. This means that the Glu1Gln exchange does not alterbinding affinity or intensity. The other differences between B-HH2 andB-HL8 are 14 FR residues, from which one or more will influence theantigen binding behavior of this antibody.

FIG. 10. Binding of the humanized anti-CD20 antibody BHL4-KV1 on Rajitarget cells. The B-HL4 construct is derived from the B-HH2 antibody byreplacing the FR1 of the B-HH2 with that of the human germ line sequenceVH1_(—)45. This construct shows greatly diminished antigen bindingcapacity, despite of having different amino acids at only threepositions within FR1. These residues are located at positions 2, 14, and30 according to Kabat numbering. Of these, position 30 seems to be themost influential position, since it is part of the Chothia definition ofCDR1.

FIG. 11. Comparison of the binding behavior between B-HH1, B-HH2, B-HH3,and the parental antibody B-ly1. The data show that all Abs show asimilar EC50 value, but the B-HH1 construct binds with a lowerintensity/stoichiometry than the variants B-HH2 and B-HH3. B-HH1 can bedistinguished from B-HH2 and B-HH3 by its partially human CDR1 and CDR2regions (Kabat definition), as well as the Ala/Thr polymorphism atposition 28 (Kabat numbering). This indicates that either position 28,the complete CDR1, and/or the complete CDR2 is important forantibody/antigen interaction.

FIG. 12. The comparison of B-HL1, B-HH1, and the B-ly1 parentalantibody. The data showed absence of any binding activity in the B-HL1construct, and about half of the binding intensity/stoichiometry ofB-HH1 compared to B-ly1. Both B-HL1, as well as B-HH1, are designedbased on acceptor frameworks derived from the human VH1 class. Amongother differences, position 71 (Kabat numbering) of the B-HL1 constructis a striking difference, indicating its putative importance for antigenbinding.

FIG. 13. Fluorocytometric analysis of the capacity of the anti-CD20antibody to its antigen. The data showed that the B-HL2 and B-HL3constructs do not display CD-20 binding activity.

FIG. 14. Apoptosis of anti-CD20 antibodies on Z-138 MCL cells.

FIGS. 15A and 15B. Apoptosis of DLBCL cell line by anti-CD20 antibodies(FIG. 15A) and apoptosis of MCL cell line by anti-CD20 antibodies (FIG.15B). Assay details: 5×10⁵ cells/well were seeded in 24-well plates(5×10⁵ cells/ml) in culture medium. 10 mg of the respective Ab, PBS forthe negative control or 5 mM Camptothecin (CPT) positive control wereadded to the wells. Samples were incubated o/n (16 h), stained withAnnV-FITC and analysed by FACS. Assay was done in triplicates.(*):Signal for PBS alone subtracted (PBS alone gave 8% and 2% AnnV+ for PR-1and Z-138 cells respectively). Antibodies used were: C2B8 (chimeric,non-glycoengineered); BHH2-KV1 (humanized, non-glycoengineered). Note:this assay does not involve any additional effector cells, just targetsplus antibody or controls.

FIGS. 16A and 16B. Target-cell killing by anti-CD20 antibodies withimmune effector cells. Assay details: B-cell depletion in normal wholeblood overnight incubation and analysis for CD19+/CD3+ by FACS (FIG.16A). ADCC using PBMCs as effectors, 4 h incubation, 25:1effector:target ratio, target-killing measured by Calcein-retentionrelative to detergent-lysis (100%) and to lysis without Ab (0%) (FIG.16B). Antibodies used: C2B8 (chimeric, non-glycoengineered form);BHH2-KV1-wt (humanized, non-glycoengineered form of BHH2-KV1);BHH2-KV1-GE (humanized, glycoengineered form of BHH2-KV1).

FIG. 17. MALDI/TOF-MS profile of PNGaseF-released Fc-oligosaccharides ofunmodified, nonglycoengineered BHH2-KV1 humanized IgG1 B-ly1 anti-humanCD20 antibody.

FIG. 18. MALDI/TOF-MS profile of PNGaseF-released Fc-oligosaccharides ofglycoengineered BHH2-KV1 g1 humanized IgG1 B-ly1 anti-human CD20antibody. Glycoengineering done by co-expression in host cells ofantibody genes and gene encoding enzyme withβ-1,4-N-acetylglucosaminyltransferase III (GnT-III) catalytic activity.

FIG. 19. MALDI/TOF-MS profile of PNGaseF-released Fc-oligosaccharides ofglycoengineered BHH2-KV1g2 humanized IgG1 B-ly1 anti-human CD20antibody. Glycoengineering done by co-expression in host cells ofantibody genes and genes encoding enzyme withβ-1,4-N-acetylglucosaminyltransferase III (GnT-III) catalytic activityand encoding enzyme with Golgi α-mannosidase II catalytic activity.

FIG. 20. Binding of non-glycoengineered and glycoengineered antibodiesto human FcgammaRIIIa receptor displayed on the surface of recombinantCHO-CD16 cells.

FIG. 21. Apoptosis of non-Fc engineered and Fc-engineered anti-CD20antibodies on Z-138 MCL cells. Assay details: 5×105 cells/well wereseeded in 24-well plates (5×105 cells/ml) in culture medium. 10 mg ofthe respective Ab, PBS for the negative control were added to the wells.Samples were incubated o/n (16 h), stained with AnnV-FITC and analysedby FACS. Assay was done in triplicates. Abs used: C2B8=rituximab(chimeric, non-glycoengineered form, same as commercial form); BHH2-KV1(humanized, non-glycoengineered—see FIG. 6 for glycosylation profile);BHH2-KV1g1 (humanized, glycoengineered—see FIG. 7 for glycosylationprofile); BHH2-KV1g2 (humanized, glycoengineered—see FIG. 8 forglycosylation profile). Note: this assay does not involve any additionaleffector cells, just targets plus antibody or controls. (*): Signal forPBS alone subtracted.

DETAILED DESCRIPTION OF THE INVENTION

Terms are used herein as generally used in the art, unless otherwisedefined as follows.

As used herein, the term antibody is intended to include whole antibodymolecules, including monoclonal, polyclonal and multispecific (e.g.,bispecific) antibodies, as well as antibody fragments having the Fcregion and retaining binding specificity, and fusion proteins thatinclude a region equivalent to the Fc region of an immunoglobulin andthat retain binding specificity. Also encompassed are humanized,primatized and chimeric antibodies.

As used herein, the term Fc region is intended to refer to a C-terminalregion of an IgG heavy chain. Although the boundaries of the Fc regionof an IgG heavy chain might vary slightly, the human IgG heavy chain Fcregion is usually defined to stretch from the amino acid residue atposition Cys226 to the carboxyl-terminus.

As used herein, the term region equivalent to the Fc region of animmunoglobulin is intended to include naturally occurring allelicvariants of the Fc region of an immunoglobulin as well as variantshaving alterations which produce substitutions, additions, or deletionsbut which do not decrease substantially the ability of theimmunoglobulin to mediate effector functions (such as antibody dependentcellular cytotoxicity). For example, one or more amino acids can bedeleted from the N-terminus or C-terminus of the Fc region of animmunoglobulin without substantial loss of biological function. Suchvariants can be selected according to general rules known in the art soas to have minimal effect on activity. (See, e.g., Bowic, J. U. et al.,Science 247:1306-10 (1990).

As used herein, the term antigen binding molecule refers in its broadestsense to a molecule that specifically binds an antigenic determinant.More specifically, an antigen binding molecule that binds CD20 is amolecule which specifically binds to a cell surface non-glycosylatedphosphoprotein of 35,000 Daltons, typically designated as the human Blymphocyte restricted differentiation antigen Bp35, commonly referred toas CD20. By “specifically binds” is meant that the binding is selectivefor the antigen and can be discriminated from unwanted or nonspecificinteractions.

As used herein, the terms fusion and chimeric, when used in reference topolypeptides such as ABMs refer to polypeptides comprising amino acidsequences derived from two or more heterologous polypeptides, such asportions of antibodies from different species. For chimeric ABMs, forexample, the non-antigen binding components may be derived from a widevariety of species, including primates such as chimpanzees and humans.The constant region of the chimeric ABM is most preferably substantiallyidentical to the constant region of a natural human antibody; thevariable region of the chimeric antibody is most preferablysubstantially identical to that of a recombinant antiCD-20 antibodyhaving the amino acid sequence of the murine B-Ly1 variable region.Humanized antibodies are a particularly preferred form of fusion orchimeric antibody.

As used herein, a polypeptide having “GnTIII activity” refers topolypeptides that are able to catalyze the addition of aN-acetylglucosamine (GlcNAc) residue in β-1-4 linkage to the n-linkedmannoside of the trimannosyl core of N-linked oligosaccharides. Thisincludes fusion polypeptides exhibiting enzymatic activity similar to,but not necessarily identical to, an activity ofβ(1,4)-N-acetylglucosaminyltransferase III, also known asβ-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC2.4.1.144), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB), as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof GnTIII, but rather substantially similar to the dose-dependence in agiven activity as compared to the GnTIII (i.e., the candidatepolypeptide will exhibit greater activity or not more than about 25-foldless and, preferably, not more than about tenfold less activity, andmost preferably, not more than about three-fold less activity relativeto the GnTIII.)

As used herein, the term variant (or analog) refers to a polypeptidediffering from a specifically recited polypeptide of the invention byamino acid insertions, deletions, and substitutions, created using,e.g., recombinant DNA techniques. Variants of the ABMs of the presentinvention include chimeric, primatized or humanized antigen bindingmolecules wherein one or several of the amino acid residues are modifiedby substitution, addition and/or deletion in such manner that does notsubstantially affect antigen (e.g., CD20) binding affinity. Guidance indetermining which amino acid residues may be replaced, added or deletedwithout abolishing activities of interest, may be found by comparing thesequence of the particular polypeptide with that of homologous peptidesand minimizing the number of amino acid sequence changes made in regionsof high homology (conserved regions) or by replacing amino acids withconsensus sequence.

Alternatively, recombinant variants encoding these same or similarpolypeptides may be synthesized or selected by making use of the“redundancy” in the genetic code. Various codon substitutions, such asthe silent changes which produce various restriction sites, may beintroduced to optimize cloning into a plasmid or viral vector orexpression in a particular prokaryotic or eukaryotic system. Mutationsin the polynucleotide sequence may be reflected in the polypeptide ordomains of other peptides added to the polypeptide to modify theproperties of any part of the polypeptide, to change characteristicssuch as ligand-binding affinities, interchain affinities, ordegradation/turnover rate.

Preferably, amino acid “substitutions” are the result of replacing oneamino acid with another amino acid having similar structural and/orchemical properties, i.e., conservative amino acid replacements.“Conservative” amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; positivelycharged (basic) amino acids include arginine, lysine, and histidine; andnegatively charged (acidic) amino acids include aspartic acid andglutamic acid. “Insertions” or “deletions” are preferably in the rangeof about 1 to 20 amino acids, more preferably 1 to 10 amino acids. Thevariation allowed may be experimentally determined by systematicallymaking insertions, deletions, or substitutions of amino acids in apolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity.

As used herein, the term humanized is used to refer to anantigen-binding molecule derived from a non-human antigen-bindingmolecule, for example, a murine antibody, that retains or substantiallyretains the antigen-binding properties of the parent molecule but whichis less immunogenic in humans. This may be achieved by various methodsincluding (a) grafting the entire non-human variable domains onto humanconstant regions to generate chimeric antibodies, (b) grafting only thenon-human CDRs onto human framework and constant regions with or withoutretention of critical framework residues (e.g., those that are importantfor retaining good antigen binding affinity or antibody functions), or(c) transplanting the entire non-human variable domains, but “cloaking”them with a human-like section by replacement of surface residues. Suchmethods are disclosed in Jones et al., Morrison et al., Proc. Natl.Acad. Sci., 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol.,44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988);Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun.,31(3):169-217 (1994), all of which are incorporated by reference intheir entirety herein. There are generally 3 complementarity determiningregions, or CDRs, (CDR1, CDR2 and CDR3) in each of the heavy and lightchain variable domains of an antibody, which are flanked by fourframework subregions (i.e., FR1, FR2, FR3, and FR4) in each of the heavyand light chain variable domains of an antibody:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A discussion of humanized antibodies canbe found, inter alia, in U.S. Pat. No. 6,632,927, and in published U.S.Application No. 2003/0175269, both of which are incorporated herein byreference in their entirety.

Similarly, as used herein, the term primatized is used to refer to anantigen-binding molecule derived from a non-primate antigen-bindingmolecule, for example, a murine antibody, that retains or substantiallyretains the antigen-binding properties of the parent molecule but whichis less immunogenic in primates.

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference, where the definitions include overlapping or subsets ofamino acid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table I as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE 1 CDR Definitions¹ Kabat Chothia AbM V_(H) CDR1 31-35 26-32 26-35V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102 V_(L)CDR1 24-34 26-32 V_(L) CDR2 50-56 50-52 V_(L) CDR3 89-97 91-96¹Numbering of all CDR definitions in Table 1 is according to thenumbering conventions set forth by Kabat et al. (see below).

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambigously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an ABM are according to the Kabatnumbering system. The sequences of the sequence listing (i.e., SEQ IDNO: 1 to SEQ ID NO:78) are not numbered according to the Kabat numberingsystem.

By a nucleic acid or polynucleotide having a nucleotide sequence atleast, for example, 95% “identical” to a reference nucleotide sequenceof the present invention, it is intended that the nucleotide sequence ofthe polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence. In otherwords, to obtain a polynucleotide having a nucleotide sequence at least95% identical to a reference nucleotide sequence, up to 5% of thenucleotides in the reference sequence may be deleted or substituted withanother nucleotide, or a number of nucleotides up to 5% of the totalnucleotides in the reference sequence may be inserted into the referencesequence. The query sequence may be the entire sequence shown in eitherFIG. 24 or FIG. 25.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence or polypeptide sequence of thepresent invention can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al., Comp. App. Biosci. 6:237-245 (1990). In a sequencealignment the query and subject sequences are both DNA sequences. An RNAsequence can be compared by converting U's to T's. The result of saidglobal sequence alignment is in percent identity. Preferred parametersused in a FASTDB alignment of DNA sequences to calculate percentidentity are: Matrix-Unitary, k-tuple=4, Mismatch Penalty=1, JoiningPenalty=30, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5,Gap Size Penalty 0.05, Window Size=500 or the length of the subjectnucleotide sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, or substituted with anotheramino acid. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a referencepolypeptide can be determined conventionally using known computerprograms. A preferred method for determining the best overall matchbetween a query sequence (a sequence of the present invention) and asubject sequence, also referred to as a global sequence alignment, canbe determined using the FASTDB computer program based on the algorithmof Brutlag et al., Comp. App. Biosci. 6:237-245 (1990). In a sequencealignment the query and subject sequences are either both nucleotidesequences or both amino acid sequences. The result of said globalsequence alignment is in percent identity. Preferred parameters used ina FASTDB amino acid alignment are: Matrix=PAM 0, k-tuple=2, MismatchPenalty=1, Joining Penalty=20, Randomization Group Length=0,CutoffScore=1, Window Size=sequence length, Gap Penalty=5, Gap SizePenalty=0.05, Window Size=500 or the length of the subject amino acidsequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to be made forthe purposes of the present invention.

As used herein, a nucleic acid that “hybridizes under stringentconditions” to a nucleic acid sequence of the invention, refers to apolynucleotide that hybridizes in an overnight incubation at 42° C. in asolution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mM sodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C.

As used herein, the term Golgi localization domain refers to the aminoacid sequence of a Golgi resident polypeptide which is responsible foranchoring the polypeptide in location within the Golgi complex.Generally, localization domains comprise amino terminal “tails” of anenzyme.

As used herein, the term effector function refers to those biologicalactivities attributable to the Fc region (a native sequence Fe region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include, but are not limited to, Fc receptorbinding affinity, antibody-dependent cellular cytotoxicity (ADCC),antibody-dependent cellular phagocytosis (ADCP), cytokine secretion,immune-complex-mediated antigen uptake by antigen-presenting cells,down-regulation of cell surface receptors, etc.

As used herein, the terms engineer, engineered engineering andglycosylation engineering are considered to include any manipulation ofthe glycosylation pattern of a naturally occurring or recombinantpolypeptide or fragment thereof. Glycosylation engineering includesmetabolic engineering of the glycosylation machinery of a cell,including genetic manipulations of the oligosaccharide synthesispathways to achieve altered glycosylation of glycoproteins expressed incells. Furthermore, glycosylation engineering includes the effects ofmutations and cell environment on glycosylation.

As used herein, the term host cell covers any kind of cellular systemwhich can be engineered to generate the polypeptides and antigen-bindingmolecules of the present invention. In one embodiment, the host cell isengineered to allow the production of an antigen binding molecule withmodified glycoforms. In a preferred embodiment, the antigen bindingmolecule is an antibody, antibody fragment, or fusion protein. Incertain embodiments, the host cells have been further manipulated toexpress increased levels of one or more polypeptides having GnTIIIactivity. Host cells include cultured cells, e.g., mammalian culturedcells, such as CHO cells, BHK cells, NSO cells, SP2/0 cells, YO myelomacells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridomacells, yeast cells, insect cells, and plant cells, to name only a few,but also cells comprised within a transgenic animal, transgenic plant orcultured plant or animal tissue.

As used herein, the term Fc-mediated cellular cytotoxicity includesantibody-dependent cellular cytotoxicity and cellular cytotoxicitymediated by a soluble Fc-fusion protein containing a human Fc-region. Itis an immune mechanism leading to the lysis of “antibody-targeted cells”by “human immune effector cells”, wherein:

The human immune effector cells are a population of leukocytes thatdisplay Fc receptors on their surface through which they bind to theFc-region of antibodies or of Fc-fusion proteins and perform effectorfunctions. Such a population may include, but is not limited to,peripheral blood mononuclear cells (PBMC) and/or natural killer (NK)cells.

The antibody-targeted cells are cells bound by the antibodies orFc-fusion proteins. The antibodies or Fc fusion-proteins bind to targetcells via the protein part N-terminal to the Fc region.

As used herein, the term increased Fe-mediated cellular cytotoxicity isdefined as either an increase in the number of “antibody-targeted cells”that are lysed in a given time, at a given concentration of antibody, orof Fc-fusion protein, in the medium surrounding the target cells, by themechanism of Fc-mediated cellular cytotoxicity defined above, and/or areduction in the concentration of antibody, or of Fc-fusion protein, inthe medium surrounding the target cells, required to achieve the lysisof a given number of “antibody-targeted cells”, in a given time, by themechanism of Fc-mediated cellular cytotoxicity. The increase inFc-mediated cellular cytotoxicity is relative to the cellularcytotoxicity mediated by the same antibody, or Fc-fusion protein,produced by the same type of host cells, using the same standardproduction, purification, formulation and storage methods, which areknown to those skilled in the art, but that has not been produced byhost cells engineered to express the glycosyltransferase GnTIII by themethods described herein.

By antibody having increased antibody dependent cellular cytotoxicity(ADCC) is meant an antibody, as that term is defined herein, havingincreased ADCC as determined by any suitable method known to those ofordinary skill in the art. One accepted in vitro ADCC assay is asfollows:

1) the assay uses target cells that are known to express the targetantigen recognized by the antigen-binding region of the antibody;

2) the assay uses human peripheral blood mononuclear cells (PBMCs),isolated from blood of a randomly chosen healthy donor, as effectorcells;

3) the assay is carried out according to following protocol:

-   -   i) the PBMCs are isolated using standard density centrifugation        procedures and are suspended at 5×10⁶ cells/ml in RPMI cell        culture medium;    -   ii) the target cells are grown by standard tissue culture        methods, harvested from the exponential growth phase with a        viability higher than 90%, washed in RPMI cell culture medium,        labeled with 100 micro-Curies of ⁵¹Cr, washed twice with cell        culture medium, and resuspended in cell culture medium at a        density of 10⁵ cells/ml;    -   iii) 100 microliters of the final target cell suspension above        are transferred to each well of a 96-well microtiter plate;    -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04        ng/ml in cell culture medium and 50 microliters of the resulting        antibody solutions are added to the target cells in the 96-well        microtiter plate, testing in triplicate various antibody        concentrations covering the whole concentration range above;    -   v) for the maximum release (MR) controls, 3 additional wells in        the plate containing the labeled target cells, receive 50        microliters of a 2% (V/V) aqueous solution of non-ionic        detergent (Nonidet, Sigma, St. Louis), instead of the antibody        solution (point iv above);    -   vi) for the spontaneous release (SR) controls, 3 additional        wells in the plate containing the labeled target cells, receive        50 microliters of RPMI cell culture medium instead of the        antibody solution (point iv above);    -   vii) the 96-well microtiter plate is then centrifuged at 50×g        for 1 minute and incubated for 1 hour at 4° C.;    -   viii) 50 microliters of the PBMC suspension (point i above) are        added to each well to yield an effector:target cell ratio of        25:1 and the plates are placed in an incubator under 5% CO₂        atmosphere at 37° C. for 4 hours;    -   ix) the cell-free supernatant from each well is harvested and        the experimentally released radioactivity (ER) is quantified        using a gamma counter,    -   x) the percentage of specific lysis is calculated for each        antibody concentration according to the formula        (ER-MR)/(MR-SR)×100, where ER is the average radioactivity        quantified (see point ix above) for that antibody concentration,        MR is the average radioactivity quantified (see point ix above)        for the MR controls (see point v above), and SR is the average        radioactivity quantified (see point ix above) for the SR        controls (see point vi above);

4) “increased ADCC” is defined as either an increase in the maximumpercentage of specific lysis observed within the antibody concentrationrange tested above, and/or a reduction in the concentration of antibodyrequired to achieve one half of the maximum percentage of specific lysisobserved within the antibody concentration range tested above. Theincrease in ADCC is relative to the ADCC, measured with the above assay,mediated by the same antibody, produced by the same type of host cells,using the same standard production, purification, formulation andstorage methods, which are known to those skilled in the art, but thathas not been produced by host cells engineered to overexpress GnTIII.

In one aspect, the present invention is related to antigen bindingmolecules having the binding specificity of the murine B-Ly1 antibody,and to the discovery that their effector functions can be enhanced byaltered glycosylation. In one embodiment, the antigen binding moleculeis a chimeric antibody. In a preferred embodiment, the invention isdirected to a chimeric antibody, or a fragment thereof, comprising theCDRs shown in FIG. 7. Specifically, in a preferred embodiment, theinvention is directed to an isolated polynucleotide comprising: (a) asequence selected from a group consisting of: SEQ ID NO.:5, SEQ ID NO.:6and SEQ ID NO.:7. (CDRs V_(H-1)); and (b) a sequence selected from agroup consisting of: SEQ ID NO.:21, SEQ ID NO.:22 and SEQ ID NO.:23.(CDRs V_(H-2)); and SEQ ID NO:24. In another preferred embodiment, theinvention is directed to an isolated polynucleotide comprising SEQ IDNO.:8, SEQ ID NO.:9 and SEQ ID NO.:10. (CDRs V_(L)). In one embodiment,any of these polynucleotides encodes a fusion polypeptide.

In another embodiment, the antigen binding molecule comprises the V_(H)domain of the murine B-Ly1 antibody shown in FIG. 1, or a variantthereof; and a non-murine polypeptide. In another preferred embodiment,the invention is directed to an antigen binding molecule comprising theV_(L) domain of the murine B-Ly1 antibody shown in FIG. 2, or a variantthereof; and a non-murine polypeptide.

In another aspect, the invention is directed to antigen bindingmolecules comprising one or more truncated CDRs of BLy-1. Such truncatedCDRs will contain, at a minimum, the specificity-determining amino acidresidues for the given CDR. By “specificity-determining residue” ismeant those residues that are directly involved in the interaction withthe antigen. In general, only about one-fifth to one-third of theresidues in a given CDR participate in binding to antigen. Thespecificity-determining residues in a particular CDR can be identifiedby, for example, computation of interatomic contacts fromthree-dimensional modeling and determination of the sequence variabilityat a given residue position in accordance with the methods described inPadlan et al., FASEB J. 9(1): 133-139 (1995), the contents of which arehereby incorporated by reference in their entirety.

Accordingly, the invention is also directed to an isolatedpolynucleotide comprising at least one complementarity determiningregion of the murine B-Ly1 antibody, or a variant or truncated formthereof containing at least the specificity-determining residues forsaid complementarity determining region, wherein said isolatedpolynucleotide encodes a fusion polypeptide. Preferably, such isolatedpolynucleotides encode a fusion polypeptide that is an antigen bindingmolecule. In one embodiment, the polynucleotide comprises threecomplementarity determining regions of the murine B-Ly1 antibody, orvariants or truncated forms thereof containing at least thespecificity-determining residues for each of said three complementaritydetermining regions. In another embodiment, the polynucleotide encodesthe entire variable region of the light or heavy chain of a chimeric(e.g., humanized) antibody. The invention is further directed to thepolypeptides encoded by such polynucleotides.

In another embodiment, the invention is directed to an antigen combiningmolecule comprising at least one complementarity determining region ofthe murine B-Ly1 antibody, or a variant or truncated form thereofcontaining at lest the specificity-determining residues for saidcomplementarity determining region, and comprising a sequence derivedfrom a heterologous polypeptide. In one embodiment, the antigen bindingmolecule comprises three complementarity determining regions of themurine B-Ly1 antibody, or variants or truncated forms thereof containingat least the specificity-determining residues for each of said threecomplementarity determining regions. In another aspect, the antigenbinding molecule comprises the variable region of an antibody light orheavy chain. In one particularly useful embodiment, the antigen bindingmolecule is a chimeric, e.g., humanized, antibody. The invention is alsodirected to methods of making such antigen binding molecules, and theuse of same in the treatment of disease, including B cell lymphomas.

It is known that several mechanism are involved in the therapeuticefficacy of anti-CD20 antibodies, including antibody dependent cellularcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and theinduction of growth arrest or apoptosis. For example, the majority ofexperimental evidence indicates that rituximab operates throughconventional effector mechanisms measured by CDC and ADCC assays.Similarly, it has been shown that the resistance of different lymphomacells to rituximab in vivo is a function of their sensitivity to CDC invitro. In contrast, the mode of action in vivo of another antibody thathas been approved for therapeutic use, B1, requires neither complementnor natural killer (NK) cell activity. Rather, the efficacy of B1 invivo is due to its ability to induce potent apoptosis.

In general, anti-CD20 monoclonal antibodies fall into two distinctcategories based on their mechanism of action in eradicating lymphomacells. Type I anti-CD20 antibodies primarily utilize complement to killtarget cells, while Type II antibodies operate by different mechanisms,primarily apoptosis. Rituximab and 1F5 are examples of Type I anti-CD20antibodies, whereas B1 is an example of a Type II antibody. See, e.g.,Cragg, M. S. and Glennie, M. J., Blood 103(7):2738-2743 (April 2004);Teeling, J. L. et al., Blood 104(6):1793-1800 (September 2004), theentire contents of which are hereby incorporated by reference.

The present invention is the first known instance in which a Type IIanti-CD20 antibody has been engineered to have increases effectorfunctions such as ADCC, while still retaining potent apoptosis ability.Accordingly, the present invention is directed to an engineered Type IIanti-CD20 antibody having increased ADCC as a result of said engineeringand without loss of substantial ability to induces apoptosis. In oneembodiment, the Type II anti-CD20 antibodies have been engineered tohave an altered pattern of glycosylation in the Fc region. In aparticular embodiment, the altered glycosylation comprises an increasedlevel of bisected complex residues in the Fc region. In anotherparticular embodiment, the altered glycosylation comprises and reducedlevel of fucose residues in the Fc region. See U.S. Pat. Appl. Pub. No.2004 0093621 to Shitara et al., the entire contents of which isincorporated by reference. In another embodiment, the Type II anti-CD20antibodies have undergone polypeptide engineering as taught in U.S. Pat.No. 6,737,056 to Presta or U.S. Pat. Appl. Pub. No. 2004 0185045(Macrogenics) or U.S. Pat. Appl. Pub. No. 2004 0132101 (Xencor), theentire contents of each of which are incorporated by reference. Theinvention is further directed to methods of making such engineered TypeII antibodies and to methods of using such antibodies in the treatmentof various B cell disorders, including B cell lymphomas.

Chimeric mouse/human antibodies have been described. See, for example,Morrison, S. L et al., PNAS 11:6851-6854 (November 1984); EuropeanPatent Publication No. 173494; Boulianna, G. L, at al., Nature 312:642(December 1984); Neubeiger, M. S. et al., Nature 314:268 (March 1985);European Patent Publication No. 125023; Tan et al., J. Immunol. 135:8564(November 1985); Sun, L. K et al., Hybridoma 5(1):517 (1986); Sahagan etal., J. Immunol. 137:1066-1074 (1986). See generally, Muron, Nature312:597 (December 1984); Dickson, Genetic Engineering News 5(3) (March1985); Marx, Science 229:455 (August 1985); and Morrison, Science229:1202-1207 (September 1985). Robinson et al., in PCT PublicationNumber WO/88104936 describe a chimeric antibody with human constantregion and murine variable region, having specificity to an epitope ofCD20; the murine portion of the chimeric antibody of the Robinsonreferences is derived from the 2H7 mouse monoclonal antibody (gamma 2b,kappa). While the reference notes that the described chimeric antibodyis a “prime candidate” for the treatment of B cell disorders, thisstatement can be viewed as no more than a suggestion to those in the artto determine whether or not this suggestion is accurate for thisparticular antibody, particularly because the reference lacks any datato support an assertion of therapeutic effectiveness, and importantly,data using higher order mammals such as primates or humans.

Methodologies for generating chimeric antibodies are available to thosein the art. For example, the light and heavy chains can be expressedseparately, using, for example, immunoglobulin light chain andimmunoglobulin heavy chains in separate plasmids, or on a single (e.g.,polycistronic) vector. These can then be purified and assembled in vitrointo complete antibodies; methodologies for accomplishing such assemblyhave been described. See, for example, Scharff, M., Harvey Lectures69:125 (1974). In vitro reaction parameters for the formation of IgGantibodies from reduced isolated light and heavy chains have also beendescribed. See, for example, Sears et al., Biochem. 16(9):2016-25(1977).

In a particularly preferred embodiment, the chimeric ABM of the presentinvention is a humanized antibody. Methods for humanizing non-humanantibodies are known in the art. For example, humanized ABMs of thepresent invention can be prepared according to the methods of U.S. Pat.No. 5,225,539 to Winter, U.S. Pat. No. 6,180,370 to Queen et al., orU.S. Pat. No. 6,632,927 to Adair et al., the entire contents of each ofwhich is hereby incorporated by reference. Preferably, a humanizedantibody has one or more amino acid residues introduced into it from asource which is non-human. These non-human amino acid residues are oftenreferred to as “import” residues, which are typically taken from an“import” variable domain. Humanization can be essentially performedfollowing the method of Winter and co-workers (Jones et al., Nature,321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);Verhoeyen et al., Science, 239:1534-1536 (1988)), by substitutinghypervariable region sequences for the corresponding sequences of ahuman antibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567) wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aretypically human antibodies in which some hypervariable region residuesand possibly some FR residues are substituted by residues from analogoussites in rodent antibodies. The subject humanized anti-CD20 antibodieswill comprise constant regions of human immunoglobulin.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework region (FR) for the humanized antibody (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901(1987)). Another method of selecting the human framework sequence is tocompare the sequence of each individual subregion of the full rodentframework (i.e., FR1, FR2, FR3, and FR4) or some combination of theindividual subregions (e.g., FR1 and FR2) against a library of knownhuman variable region sequences that correspond to that frameworksubregion (e.g., as determined by Kabat numbering), and choose the humansequence for each subregion or combination that is the closest to thatof the rodent (Leung U.S. Patent Application Publication No.2003/0040606A1, published Feb. 27, 2003). Another method uses aparticular framework region derived from the consensus sequence of allhuman antibodies of a particular subgroup of light or heavy chains. Thesame framework may be used for several different humanized antibodies(Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta etal., J. Immunol., 151:2623 (1993)).

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to a preferred method, humanizedantibodies are prepared by a process of analysis of the parentalsequences and various conceptual humanized products usingthree-dimensional models of the parental and humanized sequences.Three-dimensional immunoglobulin models are commonly available and arefamiliar to those skilled in the art. Computer programs are availablewhich illustrate and display probable three-dimensional conformationalstructures of selected candidate immunoglobulin sequences. Inspection ofthese displays permits analysis of the likely role of the residues inthe functioning of the candidate immunoglobulin sequence, i.e., theanalysis of residues that influence the ability of the candidateimmunoglobulin to bind its antigen. In this way, FR residues can beselected and combined from the recipient and import sequences so thatthe desired antibody characteristic, such as increased affinity for thetarget antigen(s), is achieved. In general, the hypervariable regionresidues are directly and most substantially involved in influencingantigen binding.

In another embodiment, the antigen binding molecules of the presentinvention are engineered to have enhanced binding affinity according to,for example, the methods disclosed in U.S. Pat. Appl. Pub. No.2004/0132066 to Balint et al., the entire contents of which are herebyincorporated by reference.

In one embodiment, the antigen binding molecule of the present inventionis conjugated to an additional moiety, such as a radiolabel or a toxin.Such conjugated ABMs can be produced by numerous methods that are wellknown in the art.

A variety of radionuclides are applicable to the present invention andthose skilled in the art are credited with the ability to readilydetermine which radionuclide is most appropriate under a variety ofcircumstances. For example, ¹³¹iodine is a well known radionuclide usedfor targeted immunotherapy. However, the clinical usefulness of¹³¹iodine can be limited by several factors including: eight-dayphysical half-life; dehalogenation of iodinated antibody both in theblood and at tumor sites; and emission characteristics (eg, large gammacomponent) which can be suboptimal for localized dose deposition intumor. With the advent of superior chelating agents, the opportunity forattaching metal chelating groups to proteins has increased theopportunities to utilize other radionuclides such as ¹¹¹indium and⁹⁰yttrium. ⁹⁰Yttrium provides several benefits for utilization inradioimmunotherapeutic applications: the 64 hour half-life of ⁹⁰yttriumis long enough to allow antibody accumulation by tumor and, unlike eg,¹³¹iodine, ⁹⁰yttrium is a pure beta emitter of high energy with noaccompanying gamma irradiation in its decay, with a range in tissue of100 to 1000 cell diameters. Furthermore, the minimal amount ofpenetrating radiation allows for outpatient administration of⁹⁰yttrium-labeled antibodies. Additionally, internalization of labeledantibody is not required for cell killing, and the local emission ofionizing radiation should be lethal for adjacent tumor cells lacking thetarget antigen.

Effective single treatment dosages (i.e., therapeutically effectiveamounts) of ⁹⁰yttrium labeled anti-CD20 antibodies range from betweenabout 5 and about 75 mCi, more preferably between about 10 and about 40mCi. Effective single treatment non-marrow ablative dosages of ¹³¹iodinelabeled anti-CD20 antibodies range from between about 5 and about 70mCi, more preferably between about 5 and about 40 mCi. Effective singletreatment ablative dosages (ie, may require autologous bone marrowtransplantation) of ¹³¹iodine labeled anti-CD20 antibodies range frombetween about 30 and about 600 mCi, more preferably between about 50 andless than about 500 mCi. In conjunction with a chimeric anti-CD20antibody, owing to the longer circulating half life vis-a-vis murineantibodies, an effective single treatment non-marrow ablative dosages of¹³¹iodine labeled chimeric anti-CD20 antibodies range from between about5 and about 40 mCi, more preferably less than about 30 mCi. Imagingcriteria for, e.g., the ¹¹¹indium label, are typically less than about 5mCi.

With respect to radiolabeled anti-CD20 antibodies, therapy therewith canalso occur using a single therapy treatment or using multipletreatments. Because of the radionuclide component, it is preferred thatprior to treatment, peripheral stem cells (“PSC”) or bone marrow (“BM”)be “harvested” for patients experiencing potentially fatal bone marrowtoxicity resulting from radiation. BM and/or PSC are harvested usingstandard techniques, and then purged and frozen for possible reinfusion.Additionally, it is most preferred that prior to treatment a diagnosticdosimetry study using a diagnostic labeled antibody (eg, using¹¹¹indium) be conducted on the patient, a purpose of which is to ensurethat the therapeutically labeled antibody (eg, using ⁹⁰yttrium) will notbecome unnecessarily “concentrated” in any normal organ or tissue.

In a preferred embodiment, the present invention is directed to anisolated polynucleotide comprising a sequence that encodes a polypeptidehaving an amino acid sequence as shown in Table 3 below. The inventionis further directed to an isolated nucleic acid comprising a sequence atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotidesequence shown in Table 2 below. In another embodiment, the invention isdirected to an isolated nucleic acid comprising a sequence that encodesa polypeptide having an amino acid sequence at least 80%, 85%, 90%, 95%,96%, 97%, 98% or 99% identical to an amino acid sequence in Table 3. Theinvention also encompasses an isolated nucleic acid comprising asequence that encodes a polypeptide having the amino acid sequence ofany of the constructs in Table 3 with conservative amino acidsubstitutions.

TABLE 2 SEQ CON- ID STRUCT NUCLEOTIDE SEQUENCE NO B-HH1CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 29GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACACCTTCAGCTATTCTTGGATGAGCTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTGG ATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACGCACAGAAATTCCAAGGAAGAGTCACAATTA CCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HH2CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 31GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACGCCTTCAGCTATTCTTGGATGAACTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTGG ATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATTA CCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HH3CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 33GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACGCCTTCAGCTATTCTTGGATGAACTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTGG ATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATTA CCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TCTGTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCAGCTAGCACC B-HH4CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 35GAAGCCTGGAGCTTCAGTGAAGGTCTCCTGCAAGG TCTCCGGATACGCGTTCAGCTATTCTTGGATGAACTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTGG ATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATTA CCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HH5CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 37GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACGCGTTCAGCTATTCTTGGATGAGCTGGGTGCGGCAGGCGCCTGGACAAGGGCTCGAGTG GATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATT ACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HH6CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 39GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACGCCTTCAGCTATTCTTGGATCAATTGGGTGCGGCAGGCGCCTGGACAAGGGCTCGAGTG GATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATT ACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HH7CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 41GAAGCCTGGGAGTTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACGCCTTCAGCTATTCTTGGATCTCGTGGGTGCGGCAGGCGCCTGGACAAGGGCTCGAGTG GATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATT ACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HH8CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 43GAAGCCTGGCGCCTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACACCTTCACATACAGCTGGATGAACTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTG GATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATT ACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HH9CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 45GAAGCCTGGCGCCTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACACCTTCAGCTATTCTTGGATGAACTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTGG ATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATTA CCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HL1CAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 47GAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACACCTTCACCTATTCTTGGATGCACTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTGG ATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACGCACAGAAATTCCAAGGAAGAGTCACAATGA CACGGGACACGTCCACTTCCACCGTCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HL2GAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 49GAAGCCTGGGGCCACCGTGAAGATCTCCTGCAAGG TGTCCGGATACACCTTCACCTATTCTTGGATGCACTGGGTGCAGCAGGCCCCTGGAAAGGGGCTCGAGTG GATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACGCAGAGAAATTCCAAGGAAGAGTCACAATC ACAGCCGACACGTCCACTGACACCGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCAACCAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HL3GAGGTGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 51GAAGCCTGGGGCCACCGTGAAGATCTCCTGCAAGG TGTCCGGATACACCTTCACCTATTCTTGGATGAACTGGGTGCAGCAGGCCCCTGGAAAGGGGCTCGAGTG GATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGAAGAGTCACAATC ACAGCCGACACGTCCACTGACACCGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGT ATTACTGTGCAACCAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HL4CAGATGCAATTGGTGCAGTCTGGCGCTGAAGTTAA 53GAAGACCGGGAGTTCAGTGAAGGTCTCCTGCAAGG CTTCCGGATACACCTTCACCTATTCTTGGATGAGCTGGGTGCGGCAGGCCCCTGGACAAGGGCTCGAGTGG ATGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACGCACAGAAATTCCAAGGAAGAGTCACAATTA CCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCAGCTAGCACC B-HL8GAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGT 55CAAGCCTGGCGGGTCCCTGCGGCTCTCCTGTGCAG CCTCTGGATTCACATTTAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAAGGGCCTCGAGTGG GTGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATTA CCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HL10CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 57ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC AGGAGCCCACTCCGAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCAAGCCTGGCGGGTCCCTGCGG CTCTCCTGTGCAGCCTCTGGATTCGCATTCAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAA GGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGC AGAGTCACAATTACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGG ACACGGCCGTGTATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCAGCTAGCGAATTCTCGAB-HL11 CAGGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGT 59CAAGCCTGGCGGGTCCCTGCGGCTCTCCTGTGCAG CCTCTGGATTCACATTTAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAAGGGCCTCGAGTGG GTGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGCAGAGTCACAATTA CCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTA TTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAACCCTGGTCACCGTCT CCTCA B-HL12CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 61ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC AGGAGCTCACTCCGAAGTGCAGCTCGTGGAGTCTGGAGCAGGCTTGGTCAAGCCTGGCGGGTCCCTGCGG CTCTCCTGCGCAGCCTCTGGATTCACATTTAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAA GGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGC AGAGTCACAATTACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGG ACACGGCCGTGTATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCAGCTAGCGAATTCTCGAB-HL13 CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 63ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC AGGAGCTCACTCCGAAGTGCAGCTCGTCGAGTCTGGAGGAGGCGTGGTCAAGCCTGGCGGGTCCCTGCGG CTCTCCTGCGCAGCCTCTGGATTCACATTTAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAA GGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGC AGAGTCACAATTACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGG ACACGGCCGTGTATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCAGCTAGCGAATTCTCGAB-HL14 CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 65ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC AGGAGCTCACTCCGAAGTGCAGCTGGTCGAGTCCGGAGGAGGCTTGAAGAAGCCTGGCGGGTCCCTGCGG CTCTCCTGCGCAGCCTCTGGATTCACATTTAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAA GGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGC AGAGTCACAATTACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGG ACACGGCCGTGTATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCAGCTAGCGAATTCTCGAB-HL15 CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 67ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC AGGAGCCCACTCCGAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCAAGCCTGGCTCTTCCCTGCGG CTCTCCTGCGCAGCCTCTGGATTCACATTTAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAA GGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGC AGAGTCACAATTACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGG ACACGGCCGTGTATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCAGCTAGCGAATTCTCGAB-HL16 CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 69ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC AGGAGCCCACTCCGAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCAAGCCTGGCGGGTCCCTGCGG GTCAGCTGCGCAGCCTCTGGATTCACATTTAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAA GGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGCGATGGGATACTGACTACAATGGGAAAATTCAAGGGC AGAGTCACAATTACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGG ACACGGCCGTGTATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCAGCTAGCGAATTCTCGAB-HL17 CGGAATTCGGCCCACCGGTGGCCACCATGGACTGG 71ACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCAC AGGAGCCCACTCCGAAGTGCAGCTGGTGGAGTCTGGAGGAGGCTTGGTCAAGCCTGGCGGGTCCCTGCGG CTCTCCTGCGCAGCCTCTGGATTCACATTTAGCTATTCTTGGATGAACTGGGTGCGGCAGGCTCCTGGAAA GGGCCTCGAGTGGGTGGGACGGATCTTTCCCGGCGATGGGGATACTGACTACAATGGGAAATTCAAGGGC AGAGTCACAATTACCGCCGACAAATCCACTAGCACAGCCTATATGGAGCTGAGCAGCCTGAGATCTGAGG ACACGGCCGTGTATTACTGTGCAAGAAATGTCTTTGATGGTTACTGGCTTGTTTACTGGGGCCAGGGAAC CCTGGTCACCGTCTCCTCAGCTAGCGAATTCTCGAVH ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGC 73 Signal AGCAGCCACAGGAGCCCACTCCSequence B-KV1 GATATCGTGATGACCCAGACTCCACTCTCCCTGCCC 75GTCACCCCTGGAGAGCCCGCCAGCATTAGCTGCAG GTCTAGCAAGAGCCTCTTGCACAGCAATGGCATCACTTATTTGTATTGGTACCTGCAAAAGCCAGGGCAG TCTCCACAGCTCCTGATTTATCAAATGTCCAACCTTGTCTCTGGCGTCCCTGACCGGTTCTCCGGATCCGGG TCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCGCTC AGAATCTAGAACTTCCTTACACCTTCGGCGGAGGGACCAAGGTGGAGATCAAACGTACGGTG VL ATGGACATGAGGGTCCCCGCTCAGCTCCTGGGCCT 77Signal CCTGCTGCTCTGGTTCCCAGGTGCCAGGTGT Sequence

TABLE 3 CONSTRUCT AMINO ACID SEQUENCE SEQ ID NO B-HH1QVQLVQSGAEVKKPGSSVKVSCKASGYTFSYSWMSWVR 30QAPGQGLEWMGRIFPGDGDTDYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS B-HH2QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWMNWV 32RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQG TLVTVSS B-HH3QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWMNWV 34RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYLCARNVFDGYWLVYWGQGT LVTVSS B-HH4QVQLVQSGAEVKKPGASVKVSCKVSGYAFSYSWMNWV 36RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQG TLVTVSS B-HH5QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWMSWV 38RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQG TLVTVSS B-HH6QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVR 40QAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS B-HH7QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWISWVR 42QAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS B-HH8QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWMNWV 44RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQG TLVTVSS B-HH9QVQLVQSGAEVKKPGASVKVSCKASGYTFSYSWMNWV 46RQAPGQGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQG TLVTVSS B-HL1QVQLVQSGAEVKKPGASVKVSCKASGYTFTYSWMHWV 48RQAPGQGLEWMGRIFPGDGDTDYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQ GTLVTVSS B-HL2EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMHWV 50QQAPGKGLEWMGRIFPGDGDTDYAEKFQGRVTITADTSTDTAYMELSSLRSEDTAVYYCATNVFDGYWLVYWGQG TLVTVSS B-HL3EVQLVQSGAEVKKPGATVKISCKVSGYTFTYSWMNWV 52QQAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATNVFDGYWLVYWGQG TLVTVSS B-HL4QMQLVQSGAEVKKTGSSVKVSCKASGYTFTYSWMSWV 54RQAPGQGLEWMGRIFPGDGDTDYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQG TLVTVSS B-HL8EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMNWVR 56QAPGKGLEWVGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGT LVTVSS B-HL10EVQLVESGGGLVKPGGSLRLSCAASGFAFSYSWMNWVR 58QAPGKGLEWVGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS B-HL11QVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMNWVR 60QAPGKGLEWVGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGT LVTVSS B-HL12EVQLVESGAGLVKPGGSLRLSCAASGFTFSYSWMNWVR 62QAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGT LVTVSS B-HL13EVQLVESGGGVVKPGGSLRLSCAASGFTFSYSWMNWVR 64QAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS B-HL14EVQLVESGGGLKKPGGSLRLSCAASGFTFSYSWMNWVR 66QAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS B-HL15EVQLVESGGGLVKPGSSLRLSCAASGFTFSYSWMNWVR 68QAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGT LVTVSS B-HL16EVQLVESGGGLVKPGGSLRVSCAASGFTFSYSWMNWVR 70QAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTL VTVSS B-HL17EVQLVESGGGLVKPGGSLRLSCAASGFTFSYSWMNWVR 72QAPGKGLEWMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGT LVTVSS VH SignalMDWTWRILFLVAAATGAHS 74 Sequence B-KV1DIVMTQTPLSLPVTPGEPASISCRSSKSLLHSNGITYLYWY 76LQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKVEIKRTV VL Signal MDMRVPAQLLGLLLLWFPGARC 78Sequence

In another preferred embodiment, the present invention is directed to anisolated polynucleotide comprising a sequence that encodes a polypeptidehaving the amino acid sequence shown in FIG. 1 or FIG. 2. The inventionis further directed to an isolated nucleic acid comprising a sequence atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to thenucleotide sequence shown in FIG. 5 or FIG. 6. In another embodiment,the invention is directed to an isolated nucleic acid comprising asequence that encodes a polypeptide having an amino acid sequence atleast 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the aminoacid sequence FIG. 5 or FIG. 6. The invention also encompasses anisolated nucleic acid comprising a sequence that encodes a polypeptidehaving the amino acid sequence of any of FIG. 1, FIG. 2, FIG. 5 or FIG.6 with conservative amino acid substitutions.

In another embodiment, the present invention is directed to anexpression vector and/or a host cell which comprise one or more isolatedpolynucleotides of the present invention.

Generally, any type of cultured cell line can be used to express the ABMof the present invention. In a preferred embodiment, CHO cells, BHKcells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myelomacells, PER cells, PER.C6 cells or hybridoma cells, other mammaliancells, yeast cells, insect cells, or plant cells are used as thebackground cell line to generate the engineered host cells of theinvention.

The therapeutic efficacy of the ABMs of the present invention can beenhanced by producing them in a host cell that further expresses apolynucleotide encoding a polypeptide having GnTIII activity. In apreferred embodiment, the polypeptide having GnTIII activity is a fusionpolypeptide comprising the Golgi localization domain of a Golgi residentpolypeptide. In another preferred embodiment, the expression of the ABMsof the present invention in a host cell that expresses a polynucleotideencoding a polypeptide having GnTIII activity results in ABMs withincreased Fc receptor binding affinity and increased effector function.Accordingly, in one embodiment, the present invention is directed to ahost cell comprising (a) an isolated nucleic acid comprising a sequenceencoding a polypeptide having GnTIII activity; and (b) an isolatedpolynucleotide encoding an ABM of the present invention, such as achimeric, primatized or humanized antibody that binds human CD20. In apreferred embodiment, the polypeptide having GnTIII activity is a fusionpolypeptide comprising the catalytic domain of GnTIII and the Golgilocalization domain is the localization domain of mannosidase II.Methods for generating such fusion polypeptides and using them toproduce antibodies with increased effector functions are disclosed inU.S. Provisional Pat. Appl. No. 60/495,142, the entire contents of whichare expressly incorporated herein by reference. In another preferredembodiment, the chimeric ABM is a chimeric antibody or a fragmentthereof, having the binding specificity of the murine B-LY1 antibody. Ina particularly preferred embodiment, the chimeric antibody comprises ahuman Fc. In another preferred embodiment, the antibody is primatized orhumanized.

In one embodiment, one or several polynucleotides encoding an ABM of thepresent invention may be expressed under the control of a constitutivepromoter or, alternately, a regulated expression system. Suitableregulated expression systems include, but are not limited to, atetracycline-regulated expression system, an ecdysone-inducibleexpression system, a lac-switch expression system, aglucocorticoid-inducible expression system, a temperature-induciblepromoter system, and a metallothionein metal-inducible expressionsystem. If several different nucleic acids encoding an ABM of thepresent invention are comprised within the host cell system, some ofthem may be expressed under the control of a constitutive promoter,while others are expressed under the control of a regulated promoter.The maximal expression level is considered to be the highest possiblelevel of stable polypeptide expression that does not have a significantadverse effect on cell growth rate, and will be determined using routineexperimentation. Expression levels are determined by methods generallyknown in the art, including Western blot analysis using an antibodyspecific for the ABM or an antibody specific for a peptide tag fused tothe ABM; and Northern blot analysis. In a further alternative, thepolynucleotide may be operatively linked to a reporter gene; theexpression levels of a chimeric ABM having substantially the samebinding specificity of the murine B-Ly1 antibody are determined bymeasuring a signal correlated with the expression level of the reportergene. The reporter gene may be transcribed together with the nucleicacid(s) encoding said fusion polypeptide as a single mRNA molecule;their respective coding sequences may be linked either by an internalribosome entry site (IRES) or by a cap-independent translation enhancer(CITE). The reporter gene may be translated together with at least onenucleic acid encoding a chimeric ABM having substantially the samebinding specificity of the murine B-Ly1 antibody such that a singlepolypeptide chain is formed. The nucleic acids encoding the AMBs of thepresent invention may be operatively linked to the reporter gene underthe control of a single promoter, such that the nucleic acid encodingthe fusion polypeptide and the reporter gene are transcribed into an RNAmolecule which is alternatively spliced into two separate messenger RNA(mRNA) molecules; one of the resulting mRNAs is translated into saidreporter protein, and the other is translated into said fusionpolypeptide.

Methods which are well known to those skilled in the art can be used toconstruct expression vectors containing the coding sequence of an ABMhaving substantially the same binding specificity of the murine B-Ly1antibody along with appropriate transcriptional/translational controlsignals. These methods include in vitro recombinant DNA techniques,synthetic techniques and in vivo recombination/genetic recombination.See, for example, the techniques described in Maniatis et al., MOLECULARCLONING A LABORATORY MANUAL, Cold Spring Harbor Laboratory, N.Y. (1989)and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, GreenePublishing Associates and Wiley Interscience, N.Y (1989).

A variety of host-expression vector systems may be utilized to expressthe coding sequence of the ABMs of the present invention. Preferably,mammalian cells are used as host cell systems transfected withrecombinant plasmid DNA or cosmid DNA expression vectors containing thecoding sequence of the protein of interest and the coding sequence ofthe fusion polypeptide. Most preferably, CHO cells, BHK cells, NSOcells, SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PERcells, PER.C6 cells or hybridoma cells, other mammalian cells, yeastcells, insect cells, or plant cells are used as host cell system. Someexamples of expression systems and selection methods are described inthe following references, and references therein: Borth et al.,Biotechnol. Bioen. 71(4):266-73 (2000-2001), in Werner et al.,Arzneimittelforschung/Drug Res. 48(8):870-80 (1998), in Andersen andKrummen, Curr. Op. Biotechnol. 13:117-123 (2002), in Chadd and Chamow,Curr. Op. Biotechnol. 12:188-194 (2001), and in Giddings, Curr. Op.Biotechnol. 12: 450-454 (2001). In alternate embodiments, othereukaryotic host cell systems may be contemplated, including yeast cellstransformed with recombinant yeast expression vectors containing thecoding sequence of an ABM of the present invention; insect cell systemsinfected with recombinant virus expression vectors (e.g., baculovirus)containing the coding sequence of a chimeric ABM having substantiallythe same binding specificity of the murine B-Ly1 antibody, plant cellsystems infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the coding sequence of the ABM of the invention; oranimal cell systems infected with recombinant virus expression vectors(e.g., adenovirus, vaccinia virus) including cell lines engineered tocontain multiple copies of the DNA encoding a chimeric ABM havingsubstantially the same binding specificity of the murine B-Ly1 antibodyeither stably amplified (CHO/dhfr) or unstably amplified indouble-minute chromosomes (e.g., murine cell lines). In one embodiment,the vector comprising the polynucleotide(s) encoding the ABM of theinvention is polycistronic. Also, in one embodiment the ABM discussedabove is an antibody or a fragment thereof. In a preferred embodiment,the ABM is a humanized antibody.

For the methods of this invention, stable expression is generallypreferred to transient expression because it typically achieves morereproducible results and also is more amenable to large-scaleproduction. Rather than using expression vectors which contain viralorigins of replication, host cells can be transformed with therespective coding nucleic acids controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows selection of cells whichhave stably integrated the plasmid into their chromosomes and grow toform foci which in turn can be cloned and expanded into cell lines.

A number of selection systems may be used, including, but not limitedto, the herpes simplex virus thymidine kinase (Wigler et al., Cell11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:2026 (1962)), andadenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980))genes, which can be employed in tk⁻, hgprt⁻ or aprt⁻ cells,respectively. Also, antimetabolite resistance can be used as the basisof selection for dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:3567 (1989); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981)); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)genes. Recently, additional selectable genes have been described, namelytrpB, which allows cells to utilize indole in place of tryptophan; hisD,which allows cells to utilize histinol in place of histidine (Hartman &Mulligan, Proc. Natl. Acad. Sci. USA 85:8047 (1988)); the glutaminesynthase system; and ODC (ornithine decarboxylase) which confersresistance to the ornithine decarboxylase inhibitor,2-(difluoromethyl)-DL-ornithine, DFMO (McConlogue, in: CurrentCommunications in Molecular Biology, Cold Spring Harbor Laboratory ed.(1987)).

The present invention is further directed to a method for modifying theglycosylation profile of the ABMs of the present invention that areproduced by a host cell, comprising expressing in said host cell anucleic acid encoding an ABM of the invention and a nucleic acidencoding a polypeptide with GnTIII activity, or a vector comprising suchnucleic acids. Preferably, the modified polypeptide is IgG or a fragmentthereof comprising the Fc region. In a particularly preferred embodimentthe ABM is a humanized antibody or a fragment thereof.

The modified ABMs produced by the host cells of the invention exhibitincreased Fc receptor binding affinity and/or increased effectorfunction as a result of the modification. In a particularly preferredembodiment the ABM is a humanized antibody or a fragment thereofcontaining the Fc region. Preferably, the increased Fc receptor bindingaffinity is increased binding to a Fcγ activating receptor, such as theFcγRIIIa receptor. The increased effector function is preferably anincrease in one or more of the following: increased antibody-dependentcellular cytotoxicity, increased antibody-dependent cellularphagocytosis (ADCP), increased cytokine secretion, increasedimmune-complex-mediated antigen uptake by antigen-presenting cells,increased Fc-mediated cellular cytotoxicity, increased binding to NKcells, increased binding to macrophages, increased binding topolymorphonuclear cells (PMNs), increased binding to monocytes,increased crosslinking of target-bound antibodies, increased directsignaling inducing apoptosis, increased dendritic cell maturation, andincreased T cell priming.

The present invention is also directed to a method for producing an ABMof the present invention, having modified oligosaccharides in a hostcell comprising (a) culturing a host cell engineered to express at leastone nucleic acid encoding a polypeptide having GnTIII activity underconditions which permit the production of an ABM according to thepresent invention, wherein said polypeptide having GnTIII activity isexpressed in an amount sufficient to modify the oligosaccharides in theFc region of said ABM produced by said host cell; and (b) isolating saidABM. In a preferred embodiment, the polypeptide having GnTIII activityis a fusion polypeptide comprising the catalytic domain of GnTIII. In aparticularly preferred embodiment, the fusion polypeptide furthercomprises the Golgi localization domain of a Golgi resident polypeptide.

Preferably, the Golgi localization domain is the localization domain ofmannosidase II or GnTI. Alternatively, the Golgi localization domain isselected from the group consisting of: the localization domain ofmannosidase I, the localization domain of GnTII, and the localizationdomain of α 1-6 core fucosyltransferase. The ABMs produced by themethods of the present invention have increased Fc receptor bindingaffinity and/or increased effector function. Preferably, the increasedeffector function is one or more of the following: increased Fc-mediatedcellular cytotoxicity (including increased antibody-dependent cellularcytotoxicity), increased antibody-dependent cellular phagocytosis(ADCP), increased cytokine secretion, increased immune-complex-mediatedantigen uptake by antigen-presenting cells, increased binding to NKcells, increased binding to macrophages, increased binding to monocytes,increased binding to polymorphonuclear cells, increased direct signalinginducing apoptosis, increased crosslinking of target-bound antibodies,increased dendritic cell maturation, or increased T cell priming. Theincreased Fc receptor binding affinity is preferably increased bindingto Fc activating receptors such as FcγRIIIa. In a particularly preferredembodiment the ABM is a humanized antibody or a fragment thereof.

In another embodiment, the present invention is directed to a chimericABM having substantially the same binding specificity of the murineB-Ly1 antibody produced by the methods of the invention which has anincreased proportion of bisected oligosaccharides in the Fc region ofsaid polypeptide. It is contemplated that such an ABM encompassesantibodies and fragments thereof comprising the Fc region. In apreferred embodiment, the ABM is a humanized antibody. In oneembodiment, the percentage of bisected oligosaccharides in the Fc regionof the ABM is at least 50%, more preferably, at least 60%, at least 70%,at least 80%, or at least 90%, and most preferably at least 90-95% ofthe total oligosaccharides. In yet another embodiment, the ABM producedby the methods of the invention has an increased proportion ofnonfucosylated oligosaccharides in the Fc region as a result of themodification of its oligosaccharides by the methods of the presentinvention. In one embodiment, the percentage of nonfucosylatedoligosaccharides is at least 50%, preferably, at least 60% to 70%, mostpreferably at least 75%. The nonfucosylated oligosaccharides may be ofthe hybrid or complex type. In a particularly preferred embodiment, theABM produced by the host cells and methods of the invention has anincreased proportion of bisected, nonfucosylated oligosaccharides in theFc region. The bisected, nonfucosylated oligosaccharides may be eitherhybrid or complex. Specifically, the methods of the present inventionmay be used to produce ABMs in which at least 15%, more preferably atleast 20%, more preferably at least 25%, more preferably at least 30%,more preferably at least 35% of the oligosaccharides in the Fc region ofthe ABM are bisected, nonfucosylated. The methods of the presentinvention may also be used to produce polypeptides in which at least15%, more preferably at least 20%, more preferably at least 25%, morepreferably at least 30%, more preferably at least 35% of theoligosaccharides in the Fe region of the polypeptide are bisected hybridnonfucosylated.

In another embodiment, the present invention is directed to a chimericABM having substantially the same binding specificity of the murineB-Ly1 antibody engineered to have increased effector function and/orincreased Fc receptor binding affinity, produced by the methods of theinvention. Preferably, the increased effector function is one or more ofthe following: increased Fc-mediated cellular cytotoxicity (includingincreased antibody-dependent cellular cytotoxicity), increasedantibody-dependent cellular phagocytosis (ADCP), increased cytokinesecretion, increased immune-complex-mediated antigen uptake byantigen-presenting cells, increased binding to NK cells, increasedbinding to macrophages, increased binding to monocytes, increasedbinding to polymorphonuclear cells, increased direct signaling inducingapoptosis, increased crosslinking of target-bound antibodies, increaseddendritic cell maturation, or increased T cell priming. In a preferredembodiment, the increased Fc receptor binding affinity is increasedbinding to a Fc activating receptor, most preferably FcγRIIIa. In oneembodiment, the ABM is an antibody, an antibody fragment containing theFc region, or a fusion protein that includes a region equivalent to theFc region of an immunoglobulin. In a particularly preferred embodiment,the ABM is a humanized antibody.

The present invention is further directed to pharmaceutical compositionscomprising the ABMs of the present invention and a pharmaceuticallyacceptable carrier.

The present invention is further directed to the use of suchpharmaceutical compositions in the method of treatment of cancer.Specifically, the present invention is directed to a method for thetreatment of cancer comprising administering a therapeutically effectiveamount of the pharmaceutical composition of the invention.

The present invention further provides methods for the generation anduse of host cell systems for the production of glycoforms of the ABMs ofthe present invention, having increased Fc receptor binding affinity,preferably increased binding to Fc activating receptors, and/or havingincreased effector functions, including antibody-dependent cellularcytotoxicity. The glycoengineering methodology that can be used with theABMs of the present invention has been described in greater detail inU.S. Pat. No. 6,602,684 and Provisional U.S. Patent Application No.60/441,307 and WO 2004/065540, the entire contents of each of which isincorporated herein by reference in its entirety. The ABMs of thepresent invention can alternatively be glycoengineered to have reducedfucose residues in the Fc region according to the techniques disclosedin EP 1 176 195 A1, the entire contents of which is incorporated byreference herein.

Generation of Cell Lines for the Production of Proteins with AlteredGlycosylation Pattern

The present invention provides host cell expression systems for thegeneration of the ABMs of the present invention having modifiedglycosylation patterns. In particular, the present invention provideshost cell systems for the generation of glycoforms of the ABMs of thepresent invention having an improved therapeutic value. Therefore, theinvention provides host cell expression systems selected or engineeredto express a polypeptide having GnTIII activity. In one embodiment, thepolypeptide having GnTIII activity is a fusion polypeptide comprisingthe Golgi localization domain of a heterologous Golgi residentpolypeptide. Specifically, such host cell expression systems may beengineered to comprise a recombinant nucleic acid molecule encoding apolypeptide having GnTIII, operatively linked to a constitutive orregulated promoter system.

In one specific embodiment, the present invention provides a host cellthat has been engineered to express at least one nucleic acid encoding afusion polypeptide having GnTIII activity and comprising the Golgilocalization domain of a heterologous Golgi resident polypeptide. In oneaspect, the host cell is engineered with a nucleic acid moleculecomprising at least one gene encoding a fusion polypeptide having GnTIIIactivity and comprising the Golgi localization domain of a heterologousGolgi resident polypeptide.

Generally, any type of cultured cell line, including the cell linesdiscussed above, can be used as a background to engineer the host celllines of the present invention. In a preferred embodiment, CHO cells,BHK cells, NSO cells, SP2/0 cells, YO myeloma cells, P3X63 mouse myelomacells, PER cells, PER.C6 cells or hybridoma cells, other mammaliancells, yeast cells, insect cells, or plant cells are used as thebackground cell line to generate the engineered host cells of theinvention.

The invention is contemplated to encompass any engineered host cellsexpressing a polypeptide having GnTIII activity, including a fusionpolypeptide that comprises the Golgi localization domain of aheterologous Golgi resident polypeptide as defined herein.

One or several nucleic acids encoding a polypeptide having GnTIIIactivity may be expressed under the control of a constitutive promoteror, alternately, a regulated expression system. Such systems are wellknown in the art, and include the systems discussed above. If severaldifferent nucleic acids encoding fusion polypeptides having GnTIIIactivity and comprising the Golgi localization domain of a heterologousGolgi resident polypeptide are comprised within the host cell system,some of them may be expressed under the control of a constitutivepromoter, while others are expressed under the control of a regulatedpromoter. Expression levels of the fusion polypeptides having GnTIIIactivity are determined by methods generally known in the art, includingWestern blot analysis, Northern blot analysis, reporter gene expressionanalysis or measurement of GnTIII activity. Alternatively, a lectin maybe employed which binds to biosynthetic products of the GnTIII, forexample, E₄-PHA lectin. Alternatively, a functional assay which measuresthe increased Fc receptor binding or increased effector functionmediated by antibodies produced by the cells engineered with the nucleicacid encoding a polypeptide with GnTIII activity may be used.

Identification of Transfectants or Transformants that Express theProtein Having a Modified Glycosylation Pattern

The host cells which contain the coding sequence of a chimeric ABMhaving substantially the same binding specificity of the murine B-Ly1antibody and which express the biologically active gene products may beidentified by at least four general approaches; (a) DNA-DNA or DNA-RNAhybridization; (b) the presence or absence of “marker” gene functions;(c) assessing the level of transcription as measured by the expressionof the respective mRNA transcripts in the host cell; and (d) detectionof the gene product as measured by immunoassay or by its biologicalactivity.

In the first approach, the presence of the coding sequence of a chimericABM having substantially the same binding specificity of the murineB-Ly1 antibody and the coding sequence of the polypeptide having GnTIIactivity can be detected by DNA-DNA or DNA-RNA hybridization usingprobes comprising nucleotide sequences that are homologous to therespective coding sequences, respectively, or portions or derivativesthereof.

In the second approach, the recombinant expression vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, resistance to methotrexate, transformationphenotype, occlusion body formation in baculovirus, etc.). For example,if the coding sequence of the ABM of the invention, or a fragmentthereof, and the coding sequence of the polypeptide having GnTIIIactivity are inserted within a marker gene sequence of the vector,recombinants containing the respective coding sequences can beidentified by the absence of the marker gene function. Alternatively, amarker gene can be placed in tandem with the coding sequences under thecontrol of the same or different promoter used to control the expressionof the coding sequences. Expression of the marker in response toinduction or selection indicates expression of the coding sequence ofthe ABM of the invention and the coding sequence of the polypeptidehaving GnTIII activity.

In the third approach, transcriptional activity for the coding region ofthe ABM of the invention, or a fragment thereof, and the coding sequenceof the polypeptide having GnTIII activity can be assessed byhybridization assays. For example, RNA can be isolated and analyzed byNorthern blot using a probe homologous to the coding sequences of theABM of the invention, or a fragment thereof, and the coding sequence ofthe polypeptide having GnTIII activity or particular portions thereof.Alternatively, total nucleic acids of the host cell may be extracted andassayed for hybridization to such probes.

In the fourth approach, the expression of the protein products can beassessed immunologically, for example by Western blots, immunoassayssuch as radioimmuno-precipitation, enzyme-linked immunoassays and thelike. The ultimate test of the success of the expression system,however, involves the detection of the biologically active geneproducts.

Generation and Use of ABMs Having Increased Effector Function IncludingAntibody-Dependent Cellular Cytotoxicity

In preferred embodiments, the present invention provides glycoforms ofchimeric ABMs having substantially the same binding specificity of themurine B-Ly1 antibody and having increased effector function includingantibody-dependent cellular cytotoxicity. Glycosylation engineering ofantibodies has been previously described. See, e.g., U.S. Pat. No.6,602,684, incorporated herein by reference in its entirety.

Clinical trials of unconjugated monoclonal antibodies (mAbs) for thetreatment of some types of cancer have recently yielded encouragingresults. Dillman, Cancer Biother. & Radiopharm. 12:223-25 (1997); Deo etal., Immunology Today 18:127 (1997). A chimeric, unconjugated IgG1 hasbeen approved for low-grade or follicular B-cell non-Hodgkin's lymphoma.Dillman, Cancer Biother. & Radiopharm. 12:223-25 (1997), while anotherunconjugated mAb, a humanized IgG1 targeting solid breast tumors, hasalso been showing promising results in phase III clinical trials. Deo etal., Immunology Today 18:127 (1997). The antigens of these two mAbs arehighly expressed in their respective tumor cells and the antibodiesmediate potent tumor destruction by effector cells in vitro and in vivo.In contrast, many other unconjugated mAbs with fine tumor specificitiescannot trigger effector functions of sufficient potency to be clinicallyuseful. Frost et al., Cancer 80:317-33 (1997); Surfus et al., J.Immunother. 19:184-91 (1996). For some of these weaker mAbs, adjunctcytokine therapy is currently being tested. Addition of cytokines canstimulate antibody-dependent cellular cytotoxicity (ADCC) by increasingthe activity and number of circulating lymphocytes. Frost et al., Cancer80:317-33 (1997); Surfus et al., J. Immunother. 19:184-91 (1996). ADCC,a lytic attack on antibody-targeted cells, is triggered upon binding ofleukocyte receptors to the constant region (Fc) of antibodies. Deo etal., Immunology Today 18:127 (1997).

A different, but complementary, approach to increase ADCC activity ofunconjugated IgG1s is to engineer the Fc region of the antibody. Proteinengineering studies have shown that FcγRs interact with the lower hingeregion of the IgG CH2 domain. Lund et al., J. Immunol. 157:4963-69(1996). However, FcγR binding also requires the presence ofoligosaccharides covalently attached at the conserved Asn 297 in the CH2region. Lund et al., J. Immunol. 157:4963-69 (1996); Wright andMorrison, Trends Biotech. 15:26-31 (1997), suggesting that eitheroligosaccharide and polypeptide both directly contribute to theinteraction site or that the oligosaccharide is required to maintain anactive CH2 polypeptide conformation. Modification of the oligosaccharidestructure can therefore be explored as a means to increase the affinityof the interaction.

An IgG molecule carries two N-linked oligosaccharides in its Fc region,one on each heavy chain. As any glycoprotein, an antibody is produced asa population of glycoforms which share the same polypeptide backbone buthave different oligosaccharides attached to the glycosylation sites. Theoligosaccharides normally found in the Fc region of serum IgG are ofcomplex bi-antennary type (Wormald et al., Biochemistry 36:130-38(1997), with a low level of terminal sialic acid and bisectingN-acetylglucosamine (GlcNAc), and a variable degree of terminalgalactosylation and core fucosylation. Some studies suggest that theminimal carbohydrate structure required for FcγR binding lies within theoligosaccharide core. Lund et al., J. Immunol. 157:4963-69 (1996)

The mouse- or hamster-derived cell lines used in industry and academiafor production of unconjugated therapeutic mAbs normally attach therequired oligosaccharide determinants to Fc sites. IgGs expressed inthese cell lines lack, however, the bisecting GlcNAc found in lowamounts in serum IgGs. Lifely et al., Glycobiology 318:813-22 (1995). Incontrast, it was recently observed that a rat myeloma-produced,humanized IgG1 (CAMPATH-1H) carried a bisecting GlcNAc in some of itsglycoforms. Lifely et al., Glycobiology 318:813-22 (1995). The ratcell-derived antibody reached a similar maximal in vitro ADCC activityas CAMPATH-1H antibodies produced in standard cell lines, but atsignificantly lower antibody concentrations.

The CAMPATH antigen is normally present at high levels on lymphomacells, and this chimeric mAb has high ADCC activity in the absence of abisecting GlcNAc. Lifely et al., Glycobiology 318:813-22 (1995). In theN-linked glycosylation pathway, a bisecting GlcNAc is added by GnTIII.Schachter, Biochem. Cell Biol. 64:163-81 (1986).

Previous studies used a single antibody-producing CHO cell line, thatwas previously engineered to express, in an externally-regulatedfashion, different levels of a cloned GnT III gene enzyme (Umana, P., etal., Nature Biotechnol. 17:176-180 (1999)). This approach establishedfor the first time a rigorous correlation between expression of GnTIIand the ADCC activity of the modified antibody. Thus, the inventioncontemplates a recombinant, chimeric antibody or a fragment thereof withthe binding specificity of the murine B-Ly1 antibody, having alteredglycosylation resulting from increased GnTIII activity. The increasedGnTIII activity results in an increase in the percentage of bisectedoligosaccharides, as well as a decrease in the percentage of fucoseresidues, in the Fc region of the ABM. This antibody, or fragmentthereof, has increased Fc receptor binding affinity and increasedeffector function. In addition, the invention is directed to antibodyfragment and fusion proteins comprising a region that is equivalent tothe Fc region of immunoglobulins.

Therapeutic Applications of ABMs Produced According to the Methods ofthe Invention.

The ABMs of the present can be used alone to target and kill tumor cellsin vivo. The ABMs can also be used in conjunction with an appropriatetherapeutic agent to treat human carcinoma. For example, the ABMs can beused in combination with standard or conventional treatment methods suchas chemotherapy, radiation therapy or can be conjugated or linked to atherapeutic drug, or toxin, as well as to a lymphokine or atumor-inhibitory growth factor, for delivery of the therapeutic agent tothe site of the carcinoma. The conjugates of the ABMs of this inventionthat are of prime importance are (1) immunotoxins (conjugates of the ABMand a cytotoxic moiety) and (2) labeled (e.g. radiolabeled,enzyme-labeled, or fluorochrome-labeled) ABMs in which the labelprovides a means for identifying immune complexes that include thelabeled ABM. The ABMs can also be used to induce lysis through thenatural complement process, and to interact with antibody dependentcytotoxic cells normally present.

The cytotoxic moiety of the immunotoxin may be a cytotoxic drug or anenzymatically active toxin of bacterial or plant origin, or anenzymatically active fragment (“A chain”) of such a toxin. Enzymaticallyactive toxins and fragments thereof used are diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaccaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, and enomycin. In anotherembodiment, the ABMs are conjugated to small molecule anticancer drugs.Conjugates of the ABM and such cytotoxic moieties are made using avariety of bifunctional protein coupling agents. Examples of suchreagents are SPDP, IT, bifunctional derivatives of imidoesters such adimethyl adipimidate HCl, active esters such as disuccinimidyl suberate,aldehydes such as glutaraldehyde, bis-azido compounds such as bis(p-azidobenzoyl) hexanediamine, bis-diazonium derivatives such asbis-(p-diazoniumbenzoyl)-ethylenediamine, diisocyanates such as tolylene2,6-diisocyanate, and bis-active fluorine compounds such as1,5-difluoro-2,4-dinitrobenzene. The lysing portion of a toxin may bejoined to the Fab fragment of the ABMs. Additional appropriate toxinsare known in the art, as evidenced in e.g., published U.S. PatentApplication No. 2002/0128448, incorporated herein by reference in itsentirety.

In one embodiment, a chimeric, glycoengineered ABM having substantiallythe same binding specificity of the murine B-Ly1 antibody, is conjugatedto ricin A chain. Most advantageously, the ricin A chain isdeglycosylated and produced through recombinant means. An advantageousmethod of making the ricin immunotoxin is described in Vitetta et al.,Science 238, 1098 (1987), hereby incorporated by reference.

When used to kill human cancer cells in vitro for diagnostic purposes,the conjugates will typically be added to the cell culture medium at aconcentration of at least about 10 nM. The formulation and mode ofadministration for in vitro use are not critical. Aqueous formulationsthat are compatible with the culture or perfusion medium will normallybe used. Cytotoxicity may be read by conventional techniques todetermine the presence or degree of cancer.

As discussed above, a cytotoxic radiopharmaceutical for treating cancermay be made by conjugating a radioactive isotope (e.g., I, Y, Pr) to achimeric, glycoengineered ABM having substantially the same bindingspecificity of the murine B-Ly1 antibody. The term “cytotoxic moiety” asused herein is intended to include such isotopes.

In another embodiment, liposomes are filled with a cytotoxic drug andthe liposomes are coated with the ABMs of the present invention. Becausethere are many CD20 molecules on the surface of the malignant B-cell,this method permits delivery of large amounts of drug to the correctcell type.

Techniques for conjugating such therapeutic agents to antibodies arewell known (see, e.g., Arnon et al., “Monoclonal Antibodies forImmunotargeting of Drugs in Cancer Therapy”, in Monoclonal Antibodiesand Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); and Thorpe et al., “The Preparation And CytotoxicProperties Of Antibody-Toxin Conjugates”, Immunol. Rev., 62:119-58(1982)).

Still other therapeutic applications for the ABMs of the inventioninclude conjugation or linkage, e.g., by recombinant DNA techniques, toan enzyme capable of converting a prodrug into a cytotoxic drug and theuse of that antibody-enzyme conjugate in combination with the prodrug toconvert the prodrug to a cytotoxic agent at the tumor site (see, e.g.,Senter et al., “Anti-Tumor Effects of Antibody-alkaline Phosphatase”,Proc. Natl. Acad Sci. USA 85:4842-46 (1988); “Enhancement of the invitro and in vivo Antitumor Activites of Phosphorylated Mitocycin C andEtoposide Derivatives by Monoclonal Antibody-Alkaline PhosphataseConjugates”. Cancer Research 49:5789-5792 (1989); and Senter,“Activation of Prodrugs by Antibody-Enzyme Conjugates: A New Approach toCancer Therapy,” FASEB J. 4:188-193 (1990)).

Still another therapeutic use for the ABMs of the invention involvesuse, either unconjugated, in the presence of complement, or as part ofan antibody-drug or antibody-toxin conjugate, to remove tumor cells fromthe bone marrow of cancer patients. According to this approach,autologous bone marrow may be purged ex vivo by treatment with theantibody and the marrow infused back into the patient [see, e.g., Ramsayet al., “Bone Marrow Purging Using Monoclonal Antibodies”, J. Clin.Immunol., 8(2):81-88 (1988)].

Furthermore, it is contemplated that the invention comprises asingle-chain immunotoxin comprising antigen binding domains that allowsubstantially the same specificity of binding as the murine B-Ly1antibody (e.g., polypeptides comprising the CDRs of the murine B-Ly1antibody) and further comprising a toxin polypeptide. The single-chainimmunotoxins of the invention may be used to treat human carcinoma invivo.

Similarly, a fusion protein comprising at least the antigen-bindingregion of an ABM of the invention joined to at least a functionallyactive portion of a second protein having anti-tumor activity, e.g., alymphokine or oncostatin, can be used to treat human carcinoma in vivo.

The present invention provides a method for selectively killing tumorcells expressing CD20. This method comprises reacting theimmunoconjugate (e.g., the immunotoxin) of the invention with said tumorcells. These tumor cells may be from a human carcinoma.

Additionally, this invention provides a method of treating carcinomas(for example, human carcinomas) in vivo. This method comprisesadministering to a subject a pharmaceutically effective amount of acomposition containing at least one of the immunoconjugates (e.g., theimmunotoxin) of the invention.

In a further aspect, the invention is directed to an improved method fortreating B-cell proliferative disorders including B-cell lymphoma, aswell as an autoimmune disease produced in whole or in part by pathogenicautoantibodies, based on B-cell depletion comprising administering atherapeutically effective amount of an ABM of the present invention to ahuman subject in need thereof. In a preferred embodiment, the ABM is aglycoengineered anti-CD20 antibody with a binding specificitysubstantially the same as that of the murine B-Ly1 antibody. In anotherpreferred embodiment the antibody is humanized. Examples of autoimmunediseases or disorders include, but are not limited to, immune-mediatedthrombocytopenias, such as acute idiopathic thrombocytopenic purpureaand chronic idiopathic thrombocytopenic purpurea, dermatomyositis,Sydenham's chorea, lupus nephritis, rheumatic fever, polyglandularsyndromes, Henoch-Schonlein purpura, post-streptococcal nephritis,erythema nodosum, Takayasu's arteritis, Addison's disease, erythemamultifonne, polyarteritis nodosa, ankylosing spondylitis, Goodpasture'ssyndrome, thromboangitis ubiterans, primary biliary cirrhosis,Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis,polymyositis/dermatomyositis, polychondritis, pamphigus vulgaris,Wegener's granulomatosis, membranous nephropathy, amyotrophic lateralsclerosis, tabes dorsalis, polymyaglia, pernicious anemia, rapidlyprogressive glomerulonephritis and fibrosing alveoolitis, inflammatoryresponses such as inflammatory skin diseases including psoriasis anddermatitis (e.g. atopic dermatitis); systemic scleroderma and sclerosis;responses associated with inflammatory bowel disease (such as Crohn'sdisease and ulcerative colitis); respiratory distress syndrome(including adult respiratory distress syndrome; ARDS); dermatitis;meningitis; encephalitis; uveitis; colitis; glomerulonephritis; allergicconditions such as eczema and asthma and other conditions involvinginfiltration of T cells and chronic inflammatory responses;atherosclerosis; leukocyte adhesion deficiency, rheumatoid arthritis;systemic lupus erythematosus (SLE); diabetes mellitus (e.g. Type 1diabetes mellitus or insulin dependent diabetes mellitus); multiplesclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergicencephalomyelitis; Sjorgen's syndrome; juvenile onset diabetes; andimmune responses associated with acute and delayed hypersensitivitymediated by cytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, polymyositis, granulomatosis and vasculitis; perniciousamenia (Addison's disease); diseases involving leukocyte diapedesis;central nervous system (CNS) inflammatory disorder, multiple organinjury syndrome; hemolytic anemia (including, but not limited tocryoglobinemia or Coombs positive anemia); myasthenia gravis;antigen-antibody complex mediated diseases; anti-glomerular basementmembrane disease; antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; pemphigoid bullous;pemphigus; autoimmune polyendocrinopathies; Reiter's disease; stiff-mansyndrome; Behcet disease; giant cell arteritis; immune complexnephritis; IgA nephropathy; IgM polyneuropathies; immunethrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc. Inthis aspect of the invention, the ABMs of the invention are used todeplete the blood of normal B-cells for an extended period.

In accordance with the practice of this invention, the subject may be ahuman, equine, porcine, bovine, murine, canine, feline, and aviansubjects. Other warm blooded animals are also included in thisinvention.

The subject invention further provides methods for inhibiting the growthof human tumor cells, treating a tumor in a subject, and treating aproliferative type disease in a subject. These methods compriseadministering to the subject an effective amount of the composition ofthe invention.

It is apparent, therefore, that the present invention encompassespharmaceutical compositions, combinations and methods for treating humancarcinomas, such as a B cell lymphoma. For example, the inventionincludes pharmaceutical compositions for use in the treatment of humancarcinomas comprising a pharmaceutically effective amount of an antibodyof the present invention and a pharmaceutically acceptable carrier.

The ABM compositions of the invention can be administered usingconventional modes of administration including, but not limited to,intravenous, intraperitoneal, oral, intralymphatic or administrationdirectly into the tumor. Intravenous administration is preferred.

In one aspect of the invention, therapeutic formulations containing theABMs of the invention are prepared for storage by mixing an antibodyhaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,and other organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride, benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

Exemplary anti-CD20 ABM formulations are described in WO98/56418,expressly incorporated herein by reference. This publication describes aliquid multidose formulation comprising 40 mg/mL rituximab, 25 mMacetate, 150 mM trehalose, 0.9% benzyl alcohol, 0.02% polysorbate 20 atpH 5.0 that has a minimum shelf life of two years storage at 2-8° C.Another anti-CD20 formulation of interest comprises 10 mg/mL rituximabin 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7mg/mL polysorbate 80, and Sterile Water for Injection, pH6.5. In thepresent invention, RITUXAN® will be substituted by an ABM of the presentinvention.

Lyophilized formulations adapted for subcutaneous administration aredescribed in WO97/04801. Such lyophilized formulations may bereconstituted with a suitable diluent to a high protein concentrationand the reconstituted formulation may be administered subcutaneously tothe mammal to be treated herein.

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to further provide a cytotoxic agent,chemotherapeutic agent, cytokine or immunosuppressive agent (e.g. onewhich acts on T cells, such as cyclosporin or an antibody that binds Tcells, e.g., one which binds LFA-1). The effective amount of such otheragents depends on the amount of antagonist present in the formulation,the type of disease or disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration mutes as used hereinbefore or about from 1 to 99% of theheretofore employed dosages.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antagonist, which matrices are inthe form of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andγethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

The compositions of the invention may be in a variety of dosage formswhich include, but are not limited to, liquid solutions or suspension,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The compositions of the invention also preferably include conventionalpharmaceutically acceptable carriers and adjuvants known in the art suchas human serum albumin, ion exchangers, alumina, lecithin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,and salts or electrolytes such as protamine sulfate.

The most effective mode of administration and dosage regimen for thepharmaceutical compositions of this invention depends upon the severityand course of the disease, the patient's health and response totreatment and the judgment of the treating physician. Accordingly, thedosages of the compositions should be titrated to the individualpatient. Nevertheless, an effective dose of the compositions of thisinvention will generally be in the range of from about 0.01 to about2000 mg/kg.

The molecules described herein may be in a variety of dosage forms whichinclude, but are not limited to, liquid solutions or suspensions,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application.

The composition comprising an ABM of the present invention will beformulated, dosed, and administered in a fashion consistent with goodmedical practice. Factors for consideration in this context include theparticular disease or disorder being treated, the particular mammalbeing treated, the clinic condition of the individual patient, the causeof the disease or disorder, the site of delivery of the agent, themethod of administration, the scheduling of administration, and otherfactors known to medical practitioners. The therapeutically effectiveamount of the antagonist to be administered will be governed by suchconsiderations.

As a general proposition, the therapeutically effective amount of theantibody administered parenterally per dose will be in the range ofabout 0.1 to 20 mg/kg of patient body weight per day, with the typicalinitial range of antagonist used being in the range of about 2 to 10mg/kg.

In a preferred embodiment, the ABM is an antibody, preferably ahumanized antibody Suitable dosages for such an unconjugated antibodyare, for example, in the range from about 20 mg/m² to about 1000 mg/m².In one embodiment, the dosage of the antibody differs from thatpresently recommended for RITUXAN®. For example, one may administer tothe patient one or more doses of substantially less than 375 mg/m² ofthe antibody, e.g., where the dose is in the range from about 20 mg/m²to about 250 mg/m², for example from about 50 mg/m² to about 200 mg/m².

Moreover, one may administer one or more initial dose(s) of the antibodyfollowed by one or more subsequent dose(s), wherein the mg/m² dose ofthe antibody in the subsequent dose(s) exceeds the mg/m² dose of theantibody in the initial dose(s). For example, the initial dose may be inthe range from about 20 mg/m² to about 250 mg/m² (e.g., from about 50mg/m² to about 200 mg/m²) and the subsequent dose may be in the rangefrom about 250 mg/m² to about 1000 mg/m².

As noted above, however, these suggested amounts of ABM are subject to agreat deal of therapeutic discretion. The key factor in selecting anappropriate dose and scheduling is the result obtained, as indicatedabove. For example, relatively higher doses may be needed initially forthe treatment of ongoing and acute diseases. To obtain the mostefficacious results, depending on the disease or disorder, theantagonist is administered as close to the first sign, diagnosis,appearance, or occurrence of the disease or disorder as possible orduring remissions of the disease or disorder.

The ABM of the present invention is administered by any suitable means,including parenteral, subcutaneous, intraperitoneal, intrapulinonary,and intranasal, and, if desired for local immunosuppressive treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antagonist may suitably beadministered by pulse infusion, e.g., with declining doses of theantagonist. Preferably the dosing is given by injections, mostpreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic.

One may administer other compounds, such as cytotoxic agents,chemotherapeutic agents, immunosuppressive agents and/or cytokines withthe antagonists herein. The combined administration includescoadministration, using separate formulations or a single pharmaceuticalformulation, and consecutive administration in either order, whereinpreferably there is a time period while both (or all) active agentssimultaneously exert their biological activities.

It would be clear that the dose of the composition of the inventionrequired to achieve cures may be further reduced with scheduleoptimization.

In accordance with the practice of the invention, the pharmaceuticalcarrier may be a lipid carrier. The lipid carrier may be a phospholipid.Further, the lipid carrier may be a fatty acid. Also, the lipid carriermay be a detergent. As used herein, a detergent is any substance thatalters the surface tension of a liquid, generally lowering it.

In one example of the invention, the detergent may be a nonionicdetergent. Examples of nonionic detergents include, but are not limitedto, polysorbate 80 (also known as Tween 80 or (polyoxyethylenesorbitanmonooleate), Brij, and Triton (for example Triton WR-1339 and TritonA-20).

Alternatively, the detergent may be an ionic detergent. An example of anionic detergent includes, but is not limited to, alkyltrimethylammoniumbromide.

Additionally, in accordance with the invention, the lipid carrier may bea liposome. As used in this application, a “liposome” is any membranebound vesicle which contains any molecules of the invention orcombinations thereof.

The examples below explain the invention in more detail. The followingpreparations and examples are given to enable those skilled in the artto more clearly understand and to practice the present invention. Thepresent invention, however, is not limited in scope by the exemplifiedembodiments, which are intended as illustrations of single aspects ofthe invention only, and methods which are functionally equivalent arewithin the scope of the invention. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingdrawings. Such modifications are intended to fall within the scope ofthe appended claims.

EXAMPLES

[NOTE: Unless otherwise specified, references to the numbering ofspecific amino acid residue positions in the following Examples areaccording to the Kabat numbering system.]

Example 1 Materials and Methods Cloning and Expression of RecombinantAntibody B-Ly1

B-Ly1 expressing hybridoma cells were grown in RPMI containing 10% FBSand 4 mM L-glutamine. 6×10⁶ cells with a viability >90% were harvestedand total RNA was isolated using a Qiagen RNAeasy midi kit. cDNAsencoding the variable light and heavy chains of B-Ly1 were amplified byRT-PCR. The RT-PCR reaction was performed using the followingconditions: 30 min 50° C. for the first strand cDNA synthesis; 15 min95° C. initial denaturation; 30 cycles of 1 min 94° C., 1 min 45° C.,1.5 min 72° C.; and a final elongation step for 10 min at 72° C. Theexpected size of the PCR products was confirmed by gel electrophoresis.The PCR products were cloned into suitable E. coli vectors and DNAsequencing confirmed that the variable light and heavy chain encodinggenes were isolated.

For construction of chimeric B-Ly1 expression vectors, synthetic signalsequences and appropriate restriction sites were fused to the variablechains by additional PCR reactions. After a final confirmation of thecorrect DNA sequence of the variable chains, they were combined with thecorresponding human IgG1 constant regions. Once the genes wereconstructed, they were cloned under control of the MPSV promoter andupstream of a synthetic polyA site, using two separate vectors, one foreach chain, resulting in the plasmids pETR1808 (heavy chain expressionvector) and pETR1813 (light chain expression vector). Each vectorcarried an EBV OriP sequence.

Chimeric B-Ly1 was produced by co-transfecting HEK293-EBNA cells withvectors pETR1808 and pETR1813 using a calcium phosphate-transfectionapproach. Exponentially growing HEK293-EBNA cells were transfected bythe calcium phosphate method. Cells were grown as adherent monolayercultures in T flasks using DMEM culture medium supplemented with 10%FCS, and were transfected when they were between 50 and 80% confluent.For the transfection of a T75 flask, 8 million cells were seeded 24hours before transfection in 14 ml DMEM culture medium supplemented withFCS (at 10% V/V final), 250 μg/ml neomycin, and cells were placed at 37°C. in an incubator with a 5% CO₂ atmosphere overnight. For each T75flask to be transfected, a solution of DNA, CaCl₂ and water was preparedby mixing 47 μg total plasmid vector DNA divided equally between thelight and heavy chain expression vectors, 235 μl of a IM CaCl₂ solution,and adding water to a final volume of 469 μl. To this solution, 469 μlof a 50 mM HEPES, 280 mM NaCl, 1.5 mM Na₂HPO₄ solution at pH 7.05 wereadded, mixed immediately for 10 sec and left to stand at roomtemperature for 20 sec. The suspension was diluted with 12 ml of DMEMsupplemented with 2% FCS, and added to the T75 in place of the existingmedium. The cells were incubated at 37° C., 5% CO₂ for about 17 to 20hours, then medium was replaced with 12 ml DMEM, 100/FCS. For theproduction of unmodified antibody “chB-Ly1”, the cells were transfectedonly with antibody expression vectors pETR1808 and pETR1813 in a 1:1ratio. For the production of the glycoengineered antibody “chB-Ly1-ge”,the cells were co-transfected with four plasmids, two for antibodyexpression (pETR1808 and pETR1813), one for a fusion GnTIII polypeptideexpression (pETR1519), and one for mannosidase 1 expression (pCLF9) at aratio of 4:4:1:1, respectively. At day 5 post-transfection, supernatantwas harvested, centrifuged for 5 min at 1200 rpm, followed by a secondcentrifugation for 10 min at 4000 rpm and kept at 4° C.

chB-Ly1 and chB-Ly1-ge were purified from culture supernatant usingthree sequential chromatographic steps, Protein A chromatography, cationexchange chromatography, and a size exclusion chromatography step on aSuperdex 200 column (Amersham Pharmacia) exchanging the buffer tophosphate buffer saline and collecting the monomeric antibody peak fromthis last step. Antibody concentration was estimated using aspectrophotometer from the absorbance at 280 nm.

Oligosaccharide Analysis

Oligosaccharides were enzymatically released from the antibodies byPNGaseF digestion, with the antibodies being either immobilized on aPVDF membrane or in solution.

The resulting digest solution containing the released oligosaccharideseither prepared directly for MALDI/TOF-MS analysis or was furtherdigested with EndoH glycosidase prior to sample preparation forMALDI/TOF-MS analysis.

Oligosaccharide Release Method for PVDF Membrane-Immobilized Antibodies

The wells of a 96-well plate made with a PVDF (Immobilon P, Millipore,Bedford, Mass.) membrane were wetted with 100 μl methanol and the liquidwas drawn through the PVDF membrane using vacuum applied to theMultiscreen vacuum manifold (Millipore, Bedford, Mass.). The PVDFmembranes were washed three times with 300 μl of water. The wells werethen washed with 50 μl RCM buffer (8M Urea, 360 mM Tris, 3.2 mM EDTA, pH8.6). Between 30-40 μg antibody was loaded in a well containing 10 μlRCM buffer. The liquid in the well was drawn through the membrane byapplying vacuum, and the membrane was subsequently washed twice with 50μl RCM buffer. The reduction of disulfide bridges was performed byaddition of 50 μl of 0.1M dithiothreitol in RCM and incubation at 37° C.for 1 h.

Following reduction, a vacuum was applied to remove the dithiothreitolsolution from the well. The wells were washed three times with 300 μlwater before performing the carboxymethylation of the cysteine residuesby addition of 50 μl 0.1M iodoacetic acid in RCM buffer and incubationat room temperature in the dark for 30 min.

After carboxymethylation, the wells were drawn with vacuum andsubsequently washed three times with 300 μl water. The PVDF membrane wasthen blocked, to prevent adsorption of the endoglycosidase, byincubating 100 μl of a 1% aqueous solution of polyvinylpyrrolidone 360at room temperature for 1 hour. The blocking reagent was then removed bygentle vacuum followed by three washes with 300 μl water.

N-linked oligosaccharides were released by addition of 2.5 mUpeptide-N-glycosydase F (recombinat N-Glycanase, GLYKO, Novato, Calif.)and 0.1 mU Sialidase (GLYKO, Novato, Calif.), to remove any potentialcharged monosaccharide residues, in a final volume of 25 μl in 20 mMNaHCO₃, pH7.0). Digestion was performed for 3 hours at 37° C.

Oligosaccharide Release Method for Antibodies in Solution

Between 40 and 50 μg of antibody were mixed with 2.5 mU of PNGaseF(Glyko, U.S.A.) in 2 mM Tris, pH7.0 in a final volume of 25 microliters,and the mix was incubated for 3 hours at 37° C.

Use of Endoglycosidase H Digestion of PNGaseF-Released Oligosaccharidesfor the Assignment of Hybrid Bisected Oligosaccharide Structures toMALDI/TOF-MS Neutral Oligosaccharide Peaks

The PNGaseF released oligosaccharides were subsequently digested withEndoglycosidase H (EC 3.2.1.96). For the EndoH digestion, 15 mU of EndoH(Roche, Switzerland) were added to the PNGaseF digest (antibody insolution method above) to give a final volume of 30 microliters, and themix was incubated for 3 hours at 37° C. EndoH cleaves between theN-acetylglucosamine residues of the chitobiose core of N-linkedoligosaccharides. The enzyme can only digest oligomannose and mosthybrid type glycans, whereas complex type oligosaccharides are nothydrolyzed.

Sample Preparation for MALDI/TOF-MS

The enzymatic digests containing the released oligosaccharides wereincubated for a further 3 h at room after the addition of acetic acid toa final concentration of 150 mM, and were subsequently passed through0.6 ml of cation exchange resin (AG50W-X8 resin, hydrogen form, 100-200mesh, BioRad, Switzerland) packed into a micro-bio-spin chromatographycolumn (BioRad, Switzerland) to remove cations and proteins. Onemicroliter of the resulting sample was applied to a stainless steeltarget plate, and mixed on the plate with 1 μl of sDHB matrix. sDHBmatrix was prepared by dissolving 2 mg of 2,5-dihydroxybenzoic acid plus0.1 mg of 5-methoxysalicylic acid in 1 ml of ethanol/10 mM aqueoussodium chloride 1:1 (v/v). The samples were air dried, 0.2 μl ethanolwas applied, and the samples were finally allowed to re-crystallizeunder air.

MALDI/TOF-MS

The MALDI-TOF mass spectrometer used to acquire the mass spectra was aVoyager Elite (Perspective Biosystems). The instrument was operated inthe linear configuration, with an acceleration of 20 kV and 80 ns delay.External calibration using oligosaccharide standards was used for massassignment of the ions. The spectra from 200 laser shots were summed toobtain the final spectrum.

Whole Blood B Cell Depletion

495 ul heparinized blood from a healthy donor was aliquoted into 5 mlpolystyrene tubes, 5 μl 100-fold concentrated antibody samples (1-1000ng/ml final concentration) or PBS only were added and the tubes wereincubated at 37°. After 24 h 50 μl blood was transferred to a fresh tubeand stained with anti-CD3-FITC, anti-CD19-PE and anti-CD45-CyChrome(Becton-Dickinson) for 15 min at room temperature in the dark. Beforeanalysis, 500 μl FACS buffer (PBS containing 2% FCS and 5 mM EDTA) wasadded to the tubes. The CD3-FITC and CD19-PE fluorescence of the bloodsamples were flowcytometrically analyzed by setting a threshold onCD45-CyChrome. B cell-depletion was determined by plotting the ratio ofCD19⁺ B cells to CD3+ T cells.

Binding of Anti-CD20 Antibodies to Raji Cells

500.000 in 180 μl FACS buffer (PBS containing 2% FCS and 5 mM EDTA) weretransferred to 5 ml polystyrene tubes and 20 ul 10 fold concentratedanti-CD20 antibody samples (1-5000 ng/ml final concentration) or PBSonly were added and the tubes were incubated at 4° C. for 30 min.Subsequently, samples were washed twice with FACS buffer and pelleted at300×g for 3 min. Supernatant was aspirated off and cells were taken upin 100 μl FACS buffer and 1 μl anti-Fc-specific F(ab′)2-FITC fragments(Jackson Immuno Research Laboratories, USA) was added and the tubes wereincubated at 4° C. for 30 min. Samples were washed twice with FACSbuffer and taken up in 500 μl of FACS buffer containing 0.5 μg/ml PI foranalysis by Flow Cytometry. Binding was determined by plotting thegeometric mean fluorescence against the antibody concentrations.

Example 2 High Homology Acceptor Approach

The high homology antibody acceptor framework search was performed byaligning the mouse B-ly1 protein sequence to a collection of humangerm-line sequences and picking that human sequence that showed thehighest sequence identity. Here, the sequence VH1_(—)10 from the VBasedatabase was chosen as the heavy chain framework acceptor sequence, andthe VK_(—)2_(—)40 sequence was chosen to be the framework acceptor forthe light chain. Onto these two acceptor frameworks, the threecomplementary determining regions (CDRs) of the mouse heavy and lightvariable domains were grafted. Since the framework 4 region is not partof the variable region of the germ line V gene, the alignment for thatposition was done individually. The JH4 region was chosen for the heavychain, and the JK4 region was chosen for the light chain. Molecularmodelling of the designed immunoglobulin domain revealed one spotpotentially requiring the murine amino acid residues instead of thehuman ones outside of the CDR. Re-introducing murine amino acid residuesinto the human framework would generate the so-called back mutations.For example, the human acceptor amino acid residue at Kabat position 27was back mutated to a tyrosine residue. Humanized antibody variants weredesigned that either included or omitted the back mutations. Thehumanized antibody light chain did not require any back mutations. Afterhaving designed the protein sequences, DNA sequences encoding theseproteins were synthesized as detailed below.

Mixed Framework Approach

In order to avoid introducing back mutations at critical amino acidresidue positions (critical to retain good antigen binding affinity orantibody functions) of the human acceptor framework, it was investigatedwhether either the whole framework region 1 (FR1), or framework regions1 (FR1) and 2 (FR2) together, could be replaced by human antibodysequences already having donor residues, or functionally equivalentones, at those important positions in the natural human germlinesequence. For this purpose, the VH frameworks 1 and 2 of the mouse Bly1sequence were aligned individually to human germ-line sequences. Heme,highest sequence identity was not important, and was not used, forchoosing acceptor frameworks, but instead matching of several criticalresidues was assumed to be more important. Those critical residuescomprise residues 24, 71, and 94 (Kabat numbering), and also thoseresidues at position 27, 28, and 30 (Kabat numbering), which lie outsideof the CDR1 definition by Kabat, but often are involved in antigenbinding. The IMGT sequence VH_(—)3_(—)15 was chosen as a suitable one.After having designed the protein sequences, DNA sequences encodingthese proteins were synthesized as detailed below. Using this approachno back mutations were required either for the light or heavy chain, inorder to retain good levels of antigen binding.

Synthesis of the Antibody Genes

After having designed the amino acid sequence of the humanized antibodyV region, the DNA sequence had to be generated. The DNA sequence data ofthe individual framework regions was found in the databases for humangerm line sequences. The DNA sequence of the CDR regions was taken fromthe corresponding murine cDNA data. With these sequences, the whole DNAsequence was virtually assembled. Having this DNA sequence data,diagnostic restriction sites were introduced in the virtual sequence, byintroducing silent mutations, creating recognition sites for restrictionendonucleases. To obtain the physical DNA chain, gene synthesis wasperformed (e.g., Wheeler et al. 1995). In this method, oligonucleotidesare designed from the genes of interest, such, that a series ofoligonucleotides is derived from the coding strand, and one other seriesis from the non-coding strand. The 3′ and 5′ ends of eacholigonucleotide (except the very first and last in the row) always showcomplementary sequences to two primers derived from the opposite strand.When putting these oligonucleotides into a reaction buffer suitable forany heat stable polymerase, and adding Mg^(2+,) dNTPs and a DNApolymerase, each oligonucleotide is extended from its 3′ end. The newlyformed 3′ end of one primer then anneals with the next primer of theopposite strand, and extending its sequence further under conditionssuitable for template dependant DNA chain elongation. The final productwas cloned into a conventional vector for propagation in E. coli.

Antibody Production

Human heavy and light chain leader sequences (for secretion) were addedupstream of the above variable region sequences and these were thenjoined upstream of human IgG1 kappa constant heavy and light chainsequences, respectively, using standard molecular biology techniques.The resulting full antibody heavy and light chain DNA sequences weresubcloned into mammalian expression vectors (one for the light chain andone for the heavy chain) under the control of the MPSV promoter andupstream of a synthetic polyA site, each vector carrying an EBV OriPsequence, as described in Example 1 above. Antibodies were produced asdescribed in Example 1 above, namely by co-transfecting HEK293-EBNA withthe mammalian antibody heavy and light chain expression vectors,harvesting the conditioned culture medium 5 to 7 days post-transfection,and purifying the secreted antibodies by Protein A affinitychromatography, followed by cation exchange chromatography and a finalsize exclusion chromatographic step to isolate pure monomeric IgG1antibodies. The antibodies were formulated in a 25 mM potassiumphosphate, 125 mM sodium chloride, 100 mM glycine solution of pH 6.7.Glycoengineered variants of the humanized antibody variants wereproduced by co-transfection of the antibody expression vectors togetherwith a GnT-III glycosyltransferase expression vectors, or together witha GnT-III expression vector plus a Golgi mannosidase II expressionvector, as described for the chimeric antibody in Example 1 above.Glycoengineered antibodies were purified and formulated as describedabove for the non-glycoengineered antibodies. The oligosaccharidesattached to the Fc region of the antibodies was analysed by MALDI/TOF-MSas described below.

Oligosaccharide Analysis

Oligosaccharide release method for antibodies in solution Between 40 and50 μg of antibody were mixed with 2.5 mU of PNGaseF (Glyko, U.S.A.) in 2mM Tris, pH7.0 in a final volume of 25 microliters, and the mix wasincubated for 3 hours at 37° C.

Sample Preparation for MALDI/TOF-MS

The enzymatic digests containing the released oligosaccharides wereincubated for a further 3 h at room temperature after the addition ofacetic acid to a final concentration of 150 mM, and were subsequentlypassed through 0.6 ml of cation exchange resin (AG50W-X8 resin, hydrogenform, 100-200 mesh, BioRad, Switzerland) packed into a micro-bio-spinchromatography column (BioRad, Switzerland) to remove cations andproteins. One microliter of the resulting sample was applied to astainless steel target plate, and mixed on the plate with 1 μl of sDHBmatrix. sDHB matrix was prepared by dissolving 2 mg of2,5-dihydroxybenzoic acid plus 0.1 mg of 5-methoxysalicylic acid in 1 mlof ethanol/10 mM aqueous sodium chloride 1:1 (v/v). The samples were airdried, 0.2 μl ethanol was applied, and the samples were finally allowedto re-crystallize under air.

MALDI/TOF-MS

The MALDI-TOF mass spectrometer used to acquire the mass spectra was aVoyager Elite (Perspective Biosystems). The instrument was operated inthe linear configuration, with an acceleration of 20 kV and 80 ns delay.External calibration using oligosaccharide standards was used for massassignment of the ions. The spectra from 200 laser shots were summed toobtain the final spectrum.

Antigen Binding Assay

The purified, monomeric humanized antibody variants were tested forbinding to human CD20 on Raji B-cell lymphoma target cells using a flowcytometry-based assay, as described for the chimeric B-ly1 antibody inExample 1 above.

Binding of Monomeric IgG1 Glycovariants to NK Cells andFc□RIIIA-Expressing CHO Cell Line

Human NK cells were isolated from freshly isolated peripheral bloodmononuclear cells (PBMC) applying a negative selection enriching forCD26- and CD56-positive cells (MACS system, Miltenyi Biotec GmbH,Bergisch Gladbach/Germany). The purity determined by CD56 expression wasbetween 88-95%. Freshly isolated NK cells were incubated in PBS withoutcalcium and magnesium ions (3×105 cells/ml) for 20 minutes at 37° C. toremove NK cell-associated IgG. Cells were incubated at 106 cells/ml atdifferent concentrations of anti-CD20 antibody (0, 0.1, 0.3, 1, 3, 10μg/ml) in PBS, 0.1% BSA. After several washes antibody binding wasdetected by incubating with 1:200 FITC-conjugated F(ab′)₂ goatanti-human, F(ab′)2 specific IgG (Jackson ImmunoReasearch, West Grove,Pa./USA) and anti-human CD56-PE (BD Biosciences, Allschwil/Switzerland).The anti-FcgammaRIIIA 3G8F(ab′)2 fragments (Ancell, Bayport, Minn./USA)were added at a concentration of 10 μg/ml to compete binding of antibodyglycovariants (3 μg/ml). The fluorescence intensity referring to thebound antibody variants was determined for CD56-positive cells on aFACSCalibur (BD Biosciences, Allschwil/Switzerland). CHO cells weretransfected by electroporation (280 V, 950 μF, 0.4 cm) with anexpression vector coding for the FcgammaRIIIA-Val158 α-chain and theγ-chain. Transfectants were selected by addition of 6 μg/m puromycin andstable clones were analyzed by FACS using 10 μlFITC-conjugated-anti-FcgammaRIII 3G8 monoclonal antibody (BDBiosciences, Allschwil/Switzerland) for 10⁶ cells. Binding of IgG1 toFcgammaRIIIA-Val158-expressing CHO cells was performed analogously tothe NK cell binding described above.

ADCC Assay

Human peripheral blood mononuclear cells (PBMC) were used as effectorcells and were prepared using Histopaque-1077 (Sigma Diagnostics Inc.,St. Louis, Mo. 63178 USA) and following essentially the manufacturer'sinstructions. In brief, venous blood was taken with heparinized syringesfrom volunteers. The blood was diluted 1:0.75-1.3 with PBS (notcontaining Ca++ or Mg++) and layered on Histopaque-1077. The gradientwas centrifuged at 400×g for 30 min at room temperature (RT) withoutbreaks. The interphase containing the PBMC was collected and washed withPBS (50 ml per cells from two gradients) and harvested by centrifugationat 300×g for 10 minutes at RT. After resuspension of the pellet withPBS, the PBMC were counted and washed a second time by centrifugation at200×g for 10 minutes at RT. The cells were then resuspended in theappropriate medium for the subsequent procedures.

The effector to target ratio used for the ADCC assays was 25:1 and 10:1for PBMC and NK cells, respectively. The effector cells were prepared inAIM-V medium at the appropriate concentration in order to add 50 μl perwell of round bottom 96 well plates. Target cells were human B lymphomacells (e.g., Raji cells) grown in DMEM containing 10% FCS. Target cellswere washed in PBS, counted and resuspended in AIM-V at 0.3 million perml in order to add 30′000 cells in 100 μl per microwell. Antibodies werediluted in AIM-V, added in 50 μl to the pre-plated target cells andallowed to bind to the targets for 10 minutes at RT. Then the effectorcells were added and the plate was incubated for 4 hours at 37° C. in ahumified atmosphere containing 5% CO₂. Killing of target cells wasassessed by measurement of lactate dehydrogenase (LDH) release fromdamaged cells using the Cytotoxicity Detection kit (Roche Diagnostics,Rotkreuz, Switzerland). After the 4-hour incubation the plates werecentrifuged at 800×g. 100 μl supernatant from each well was transferredto a new transparent flat bottom 96 well plate. 100 μl color substratebuffer from the kit were added per well. The Vmax values of the colorreaction were determined in an ELISA reader at 490 nm for at least 10min using SOFTmax PRO software (Molecular Devices, Sunnyvale, Calif.94089, USA). Spontaneous LDH release was measured from wells containingonly target and effector cells but no antibodies. Maximal release wasdetermined from wells containing only target cells and 1% Triton X-100.Percentage of specific antibody-mediated killing was calculated asfollows: ((x−SR)/(MR−SR)*100, where x is the mean of Vmax at a specificantibody concentration, SR is the mean of Vmax of the spontaneousrelease and MR is the mean of Vmax of the maximal release.

Complement Dependent Cytotoxicity Assay

Target cells were counted, washed with PBS, resuspended in AIM-V(Invitrogen) at 1 million cells per ml. 50 μl cells were plated per wellin a flat bottom 96 well plate. Antibody dilutions were prepared inAIM-V and added in 50 μl to the cells. Antibodies were allowed to bindto the cells for 10 minutes at room temperature. Human serum complement(Quidel) was freshly thawed, diluted 3-fold with AIM-V and added in 50μl to the wells. Rabbit complement (Cedarlane Laboratories) was preparedas described by the manufacturer, diluted 3-fold with AIM-V and added in50 μl to the wells. As a control, complement sources were heated for 30min at 56° C. before addition to the assay.

The assay plates were incubated for 2 h at 37° C. Killing of cells wasdetermined by measuring LDH release. Briefly, the plates werecentrifuged at 300×g for 3 min. 50 μl supernatant per well weretransferred to a new 96 well plate and 50 μl of the assay reagent fromthe Cytotoxicity Kit (Roche) were added. A kinetic measurement with theELISA reader determined the Vmax corresponding with LDH concentration inthe supernatant. Maximal release was determined by incubating the cellsin presence of 1% Trition X-100.

Whole Blood B-Cell Depletion Assay

Normal B-cell depletion in whole blood by the anti-CD20 antibodies wascarried out as described in Example 1 above.

Apoptosis Assay

The apoptotic potency of the antibodies was assayed by incubating theantibody at 10 μg/ml (saturating conditions in respect to antigenbinding) with the target cells (at a target cell concentration of 5×105cells/ml) overnight (16-24 h). Samples were stained with AnnV-FITC andanalyzed by FACS. Assay was done in triplicates.

Detection is performed by flow cytometry by following the appearance ofapoptotic markers like annexin V and phosphatidy serine. Negativecontrol (no apoptosis induced) does not contain any antibody, but onlyphosphate buffered saline. Positive control (maximal apoptosis) contains5 micromolar of the strong apoptosis inducer Camptothecin (CPT).

Results and Discussion

Comparison of the binding to human CD20 antigen of antibody variantsB-HH1, B-HH2, B-HH3, either complexed with the chimeric B-ly1 lightchain (mVL, as described in Example 1 above) or with the humanized B-ly1light chain (KV1), and the parental, chimeric antibody chB-ly1(described in Example 1 above) shows that all antibodies have a similarEC50 value, but the B-HH1 construct binds with a lowerintensity/stoichiometry than the variants B-HH2 and B-HH3 (FIG. 11).B-HH1 can be distinguished from B-HH2 and B-HH3 by its partially humanCDR1 and CDR2 regions (Kabat definition), as well as the Ala/Thrpolymorphism at position 28 (Kabat numbering). This indicates thateither position 28, the complete CDR1, and/or the complete CDR2 areimportant for antibody/antigen interaction.

The comparison of the B-HL1, B-HH1, and the chimeric chB-ly1 parentalantibody showed absence of any binding activity in the B-HL1 construct,and about half of the binding intensity/stoichiometry of the B-HH1compared to B-ly1 (FIG. 12). Both the B-HL1 as well as the B-HH1 aredesigned based on acceptor frameworks derived from the human VH1 class.Among other differences, position 71 (Kabat numbering; Kabat position 71corresponds to position 72 of SEQ ID NO:48) of the B-HL1 construct is astriking difference, indicating its putative importance for antigenbinding.

When comparing the antigen binding data of FIGS. 9 to 13, the BHH2-KV1,BHL8-KV1, and BHL11-KV1 variants show the best binding affinity, amongthe different humanized antibody variants tested, to human CD20 on thesurface of human cells. The differences between B-HH2, on one hand, andB-HL8 and B-HL11 on the other hand are located in the FR1 and FR2regions only, with all three CDRs being identical (compare, e.g., SEQ IDNOs: 32, 56, and 60, which are not numbered according to Kabat, butwhose Kabat numbering can be readily determined by one of ordinaryskill). B-HL8 and B-HL11 have their FR1 and FR2 sequences derived fromthe human VH3 class, whereas the complete B-HH2 framework is human VH1derived. B-HL11 is a derivative of B-HL8 with the single mutationGlu1Gln (position 1 is the same in both Kabat numbering and theconventional numbering system used in the sequence listing), with Ginbeing the amino acid residue in the B-HH2 construct. This means thatGlu1Gln exchange does not alter binding affinity nor intensity. Theother differences between B-HH2 and B-HL8 are 14 framework residues, ofwhich one or more will influence the antigen binding behavior of thisantibody.

The B-HL4 construct is derived from the B-HH2 antibody by replacing theFR1 of the B-HH2 with that of the human germ line sequence VH1_(—)45.This construct shows greatly diminished antigen binding capacity,despite having different amino acids at only three positions within FR1.These residues are located at positions 2, 14, and 30 (Kabat numbering).Of these, position 30 could be an influential position, since it is partof the Chothia definition of CDR1. Overall analysis of all the bindingcurves from FIGS. 9 to 13 indicates that the following humanized B-ly1heavy chain residues (Kabat numbering) are important for binding toCD20: N35 (end of Kabat CDR1), full Kabat CDR1, full Kabat CDR2 and fullKabat CDR3, residues A71 and R94 (in this case R94 cannot be replaced bya threonine) and Y27. A28 and S30 also contribute to a lesser extent. Inaddition, Kabat CDR3 and all canonical residues are important forantigen binding. No back mutations were introduced in the humanizedlight chain, which had the full Kabat CDR1, CDR2 and CDR3 grafted. Ininduction of apoptosis (FIGS. 14, 15 and 21), the most potent variantwas humanized B-ly1 variant BHH2-KV1 (even more potent than the originalchB-ly1 and a lot more potent than an antibody with a sequence identicalto rituximab, C2B8). Other humanized variants (derivatives of BHL8) thatcan recover the increased apoptosis are: B-HL12 to B-HL17 (see Table)and BHH8 (mixed frameworks) and BHH9 (“mixed frameworks” with one backmutation, S30T). Positions 9 and 48 (Kabat numbering) can contact theantigen. Variants BHH4 to BHH7 are other humanized B-ly1 variants thatdo not introduce additional non-human sequences.

Important properties of the humanized B-ly1 antibody are that it is atype II anti-CD20 antibody as defined in Cragg. M. S. and Glennic, M.J., Blood 103(7):2738-2743 (April 2004). It therefore did not induce,upon binding to CD20, any significant resistance to non-ionic detergentextraction of CD20 from the surface of CD20+ human cells, using theassay described for this purposes in Polyak, M. J. and Deans, J. P.,Blood 99(9):3256-3262 (2002). It certainly induced significantly lessresistance to non-ionic detergent extraction of CD20 than the C2B8antibody does (another anti-CD20 antibody with identical sequence torituximab, (See U.S. Pat. Pub. No. 2003 0003097 to Reff). As expected ofa type II anti-CD20 antibody, the humanized B-ly1 did not have anysignificant complement mediated lysis activity and certainly a lotcomplement mediated lysis activity than the anti-CD20 antibody C2B8(chimeric IgG1 with identical sequence to rituximab). Another importantproperty of the humanized B-ly1 antibody was that it was very potent inthe homotypic aggregation assay. In this assay CD20-positive humancells, Daudi cells, were incubated in cell culture medium for up to 24hours at 37° C. in a 5% CO2 atmosphere in a mammalian cell incubator asdescribed in detail in (Deans reference), with the antibody at aconcentration of 1 microgram per ml and in parallel at a concentrationof 5 micrograms per ml. As a comparison, control, parallel incubation ofthe cells were done under identical conditions but using the anti-CD20antibody C2B8. At different time points, including 8 hours and 24 hoursof incubation, the cells were inspected visually using a microscope. Itwas found that the humanized B-ly1 antibody led to strong homotypicaggregation, with aggregates being significantly larger that thoseinduced by addition of the C2B8 control antibody. In addition, andconsistent with the antibody being anti-CD20 type 1, it induced higherlevels of apoptosis when CD20-positive human cells were incubated withthe humanized B-ly1 antibody, relative to a control under identicalconditions using the C2B8 chimeric IgG1 antibody with identical sequenceto rituximab.

Glycoengineered variants of the humanized antibodies were produced byco-expression of GnTIII glycosyltransferase, together with the antibodygenes, in mammalian cells. This led to an increase in the fraction ofnon-fucosylated oligosaccharides attached to the Fc region of theantibodies, including bisected non-fucosylated oligosaccharides, as hasbeen described in WO 2004/065540 (FIGS. 17-19). The glycoengineeredantibodies had significantly higher levels of binding to humanFcgammaRIII receptors (FIG. 20) and ADCC activity as well (FIG. 16),relative to the non-glycoengineered antibody and relative to the C2B8antibody. The humanized B-ly1 antibody was also more potent at inducinghuman B-cell depletion in a whole blood assay (FIG. 16) than the controlC2B8 antibody. This was true both for the non-glycoengineered B-ly1antibody and for the glycoengineered version of it. The glycoengineeredantibody was approximately 1000-fold more potent than the C2B8 controlanti-CD20 antibody in depleting B-cells in the whole blood assay. Thiscomparison is important both for the non-glycoengineered and for theglycoengineered humanized forms of B-ly1 antibody, because it showedthat in assays that combined Fc receptor-dependent activities, such asADCC, plus complement mediated lysis, plus induction of apoptosis, thatboth forms of B-ly1 were significantly more potent that C2B8, althoughboth forms of B-ly1 have dramatically lower complement mediated lysisactivity. The ADCC, Fc receptor-dependent cell killing activities andapoptosis induction were present in this superior activity of thehumanized B-ly1 antibody variants. Furthermore, in the apoptosis assay,both the glycoengineered and non-glycoengineered forms of this type IIanti-CD20 antibody were potent, with the Fc-engineered variants withincreased binding affinity to Fcgamma receptors being even more potentin apoptosis induction than the non-Fc-engineered variant, and with allvariants being significantly more potent than the control antibody C2B8.The exact mechanism for enhanced homotypic aggregation and induction ofapoptopsis mediated by type II anti-CD20 antibodies is not known andconcomitant binding to other molecules on the surface of CD20-positivecells, such as Fc gamma receptors, can influence this importantproperty. It was therefore important to demonstrate that anti-CD20antibodies of type II that have been engineered in their Fc region forincreased binding affinity to Fc gamma receptors, including FcgammaRIIIand with an associated increase in ADCC activity, were still able toinduce strong apoptosis, even higher than the non-Fc-engineered, andhomotypic aggregation. Apoptopsis induction is important as in vivo, asthere are locations in the body where the target CD20-positive cells canbe found, but were access to FcgammaRIII-positive cells is moredifficult than in blood, such locations are, for example, lymph nodes.In those locations, the induction of apoptosis by the anti-CD20 antibodyitself can be crucial for good efficacy of the anti-CD20 antibodytherapy in humans, both for the treatment of haematological malignanciessuch as non-Hodgkins lymphomas and B-cell chronic lymphocytic leukaemia,and for the treatment of autoimmune diseases such as rheumatoidarthritis and lupus via a B-cell depletion approach. The increasedbinding affinity to FcgammaRIII and higher ADCC of the humanized,Fc-engineered type II anti-CD20 antibody can also be a very importantattribute for such therapies. Finally, the reduced or negligiblecomplement mediated lysis activity of this type II anti-CD20 antibodies,including humanized and Fc-engineered variants, can also be importanthigher complement activation by anti-CD20 antibodies has been correlatedwith increased, undesirable side-effects.

1-73. (canceled) 74: A host cell engineered to express at least onenucleic acid encoding a polypeptide havingβ(1,4)-N-acetylglucosaminyltransferase III activity in an amountsufficient to modify the oligosaccharides in the Fc region of apolypeptide produced by said host cell, wherein said polypeptide is anantigen binding molecule comprising a sequence derived from the muringB-Ly1 antibody and a sequence from a heterologous polypeptide. 75: Thehost cell of claim 74, wherein said polypeptide havingβ(1,4)-N-acetylglucosaminyltransferase III activity is a fusionpolypeptide. 76: The host cell of claim 74, wherein said antigen bindingmolecule is an antibody. 77: The host cell of claim 74, wherein saidantigen binding molecule is an antibody fragment. 78: The host cell ofclaim 74, wherein said antigen binding molecule comprises a regionequivalent to the Fc region of a human IgG. 79: The host cell of claim74, wherein said antigen binding molecule produced by said host cellexhibits increased Fc receptor binding affinity as a result of saidmodification. 80: The host cell of claim 74, wherein said antibodyproduced by said host cell exhibits increased effector function as aresult of said modification. 81: The host cell according to claim 75,wherein said fusion polypeptide comprises the catalytic domain ofβ(1,4)-N-acetylglucosaminyltransferase III. 82: The host cell accordingto claim 75, wherein said fusion polypeptide further comprises the Golgilocalization domain of a heterologous Golgi resident polypeptide. 83:The host cell according to claim 82, wherein said Golgi localizationdomain is the localization domain of mannosidase II. 84: The host cellaccording to claim 82, wherein said Golgi localization domain is thelocalization domain of β(1,2)-N-acetylglucosaminyltransferase I. 85: Thehost cell according to claim 82, wherein said Golgi localization domainis the localization domain of β(1,2)-N-acetylglucosaminyltransferase II.86: The host cell according to claim 82, wherein said Golgi localizationdomain is the localization domain of mannosidase I. 87: The host cellaccording to claim 82, wherein said Golgi localization domain is thelocalization domain of α1-6 core fucosyltransferase. 88: The host cellaccording to claim 80, wherein said increased effector function isincreased Fc-mediated cellular cytotoxicity. 89: The host cell accordingto claim 80, wherein said increased effector function is increasedbinding to NK cells. 90: The host cell according to claim 80, whereinsaid increased effector function is increased binding to macrophages.91: The host cell according to claim 80, wherein said increased effectorfunction is increased binding to polymorphonuclear cells. 92: The hostcell according to claim 80, wherein said increased effector function isincreased binding to monocytes. 93: The host cell according to claim 80,wherein said increased effector function is increased direct signalinginducing apoptosis. 94: The host cell according to claim 80, whereinsaid increased effector function is increased dendritic cell maturation.95: The host cell according to claim 80, wherein said increased effectorfunction is increased T cell priming. 96: The host cell according toclaim 79, wherein said Fc receptor is Fcγ activating receptor. 97: Thehost cell according to claim 79, wherein said Fc receptor is FcγRIIIAreceptor. 98: The host cell according to claim 74, wherein said hostcell is a CHO cell, a BHK cell, a NSO cell, a SP2/0 cell, a YO myelomacell, a P3X63 mouse myeloma cell, a PER cell, a PER.C6 cell or ahybridoma cell. 99: The host cell of claim 74, further comprising atleast one transfected polynucleotide encoding a polypeptide derived fromthe murine B-Ly1 antibody and a sequence encoding a region equivalent tothe Fc region of a human immunoglobulin. 100: The host cell of claim 74,wherein said at least one nucleic acid encoding a polypeptide havingβ(1,4)-N-acetylglucosaminyltransferase III activity is operably linkedto a constitutive promoter element. 101: The host cell of claim 100,wherein said polypeptide havingbeta(1,4)-N-acetylglucosaminyltransferase III activity is a fusionpolypeptide. 102-259. (canceled)